Genes and enzymes involved in the microbial degradation of pentachlorophenol

ABSTRACT

Three purified proteins, PcpA, PcpB and PcpC, are disclosed. These proteins are involved in the breakdown of pentachlorophenol and other halogenated phenols by the bacterium Flavobacterium sp. Strain ATCC 39723. Three cloned genes, pcpA, pcpB and pcpC, which encode these proteins are also disclosed. The pure proteins and cloned genes can be used in bioremediation applications.

The government has rights in this invention, which was funded in part by grant R-815250-01-0 from the U.S. Environmental Protection Agency.

This application is a continuation in part of U.S. patent application Ser. No. 07/856,015, filed Mar. 23, 1992.

FIELD OF THE INVENTION

The present invention concerns genes and purified enzymes involved in the microbial degradation of halogenated phenols.

BACKGROUND OF THE INVENTION

General Discussion of the Background

Halogenated aromatic compounds have been produced industrially on a large scale for several decades. Brominated aromatic compounds have found use as flame retardants, and fluorinated and iodinated aromatic compounds are components of pharmaceutical agents. The chlorinated aromatics have been widely used as pesticides (for example, 2,4-dichlorophenoxyacetic acid and pentachlorophenol) and in industrial settings (for example, polychlorinated biphenyls are employed in electrical equipment and as hydraulic fluids). Typically, these compounds are highly chemically inert, hydrophobic and toxic. Their widespread distribution in the environment is therefore of great concern.

Halogenated phenols comprise a significant subgroup of the halogenated aromatics. The halogenated phenols include pentachlorophenol and its salt, sodium pentachlorophenol. These two compounds (hereinafter referred to collectively as PCP) are among the most widely distributed biocides used in the United States. PCP is an inhibitor of oxidative phosphorylation and, as such, is lethal to a wide variety of organisms, including both plants and animals. PCP is primarily employed by the wood preserving industry as a fungicide and pesticide and is also used in products such as herbicides and disinfectants.

PCP is highly toxic and tends to persist in the environment and within food chains. The adverse effects of low-level PCP contamination on organisms such as fish, shrimp, oysters, clams, and rats are well documented and are reviewed in Rao (1977). A number of studies conclude that contamination of tissues of human populations with PCP at a level of 10 to 20 parts per billion is average for industrialized societies (Rao, 1977). PCP is thought to be mutagenic or at least co-mutagenic, and human exposure to PCP poses significant health hazards.

Removal of PCP and other halogenated aromatics from contaminated environments is therefore an issue of great concern. Methods such as the use of activated charcoal to decontaminate water are currently employed, and efforts have been directed towards the development of bioremediation approaches. Several reports have documented the ability of mixed microbial populations to degrade PCP and other halogenated aromatics. These microbes may be used in combination with existing bioremediation processes and techniques to remove PCP and other halogenated aromatics from contaminated water supplies and soils. Suitable bioremediation techniques include those disclosed in several patents, including U.S. Pat. No. 4,713,340 to Crawford, and East German patent No. 3,601,979 to Debus and Rohde, which describe methods for inoculating bacteria into contaminated environments or materials, including soils and water, and providing conditions under which the inoculated bacterial populations break down the contaminating halogenated aromatics. Another mode of use for such bacteria is disclosed in Japanese patent No. 49094569 to Nishimura in which a crude enzyme extract from a gram-negative bacillus is used to degrade polychlorinated biphenyl.

Further investigations have led to the isolation and characterization of pure strains of bacteria able to degrade halogenated aromatics. In particular, several bacterial strains have been isolated that are capable of breaking down PCP into cellular metabolites. These strains include Arthrobacter sp. Strain ATCC 33790 (Schenk et al., 1989), Rhodococcus chlorophenolicus PCP-I (DFN 43826) (Apajalahti and Salkinoja-Salonen, 1987), and Flavobacterium sp. Strain ATCC 39723 as described in U.S. Pat. No. 4,713,340 to Crawford.

The mechanisms by which such microorganisms degrade PCP and other halogenated aromatics are not completely understood. Proposed mechanisms of biodegradation of halogenated aromatics are discussed in Commandeur and Parsons (1990). Microbial metabolism of PCP is addressed in Reiner et al. (1978), and the transformation of PCP under environmental conditions including microbial breakdown has been reviewed by Engelhardt et al. (1986). Based on the isolation of metabolites extracted from culture media, various degradation pathways have been proposed. These pathways have in common the step by step dechlorination of PCP to less chlorinated compounds after which the aromatic ring is cleaved.

Studies of cell extracts from Rhodococcus chlorophenolicus have proposed that the initial step of PCP breakdown is hydroxylation to yield tetrachlorohydroquinone (Apajalahti and Salkinoja-Salonen, 1987a) which is subsequently converted to dichlorotrihydroxybenzene by a reaction involving both hydrolytic and reductive dechlorinations (Apajalahti and Salkinoja-Salonen, 1987b). Further dechlorinations then yield 1,2,4 trihydroxybenzene. Trihydrochloroquinone was found to be degraded only very slowly, suggesting that it is not an intermediate in the pathway.

A pathway involving initial hydrolytic dechlorination in the para position of PCP to form 2,3,5,6-tetrachloro-p-hydroquinone (TeCH) and further reductive dechlorinations has been proposed to be responsible for the degradation of PCP by the aerobic Flavobacterium sp. Strain ATCC 39723 (Steiert and Crawford, 1986). This strain is also able to degrade and dechlorinate a range of di, tri and tetra-chlorophenols (Steiert et al., 1987). Chlorophenols with chlorine substituents in both ortho (2 and 6) positions were degraded most readily. Of these, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol and PCP were inducers of the complete PCP degradation pathway.

The microbial breakdown of PCP is thus generally considered to proceed by hydroxylation and dechlorination. The microbes exhibiting PCP breakdown activity must produce enzymes which are able to cleave the carbon-chloride bonds in PCP. However, these and other proteins involved in PCP breakdown have been neither characterized nor purified. Similarly, the genes encoding these enzymes have yet to be identified and cloned.

It is an object of this invention to characterize the process of bacterial PCP breakdown and by so doing to provide information and materials of utility for novel bioremediation technologies.

Specifically, it is an object of this invention to identify and purify enzymes involved in the bacterial breakdown of PCP.

It is another specific object of this invention to identify and clone the genes encoding such enzymes.

It is another object of this invention to provide recombinant DNA vectors which contain the enzymes involved in the bacterial breakdown of PCP, which vectors are capable of directing the expression of these enzymes in heterologous bacterial hosts.

It is a further object of this invention to provide bacteria which are stably transformed with these vectors and which express one or more of the enzymes involved in the bacterial breakdown of PCP.

These and other objects of the invention will be understood more clearly by reference to the following detailed description.

SUMMARY OF THE INVENTION

The foregoing objects have been achieved by the identification and purification of three novel proteins which are present in the PCP degrading bacterium 5 Flavobacterium sp. Strain ATCC 39723 and which are involved in the breakdown of PCP by this organism. These purified proteins are designated PcpA, PcpB and PcpC.

The current invention also includes novel, cloned DNA molecules which include the genes encoding these three proteins. The cloned genes which encode PcpA, PcpB and PcpC are designated pcpA, pcpB and pcpC, respectively.

More specifically, a protein which is shown to be found in the periplasmic space of the Flavobacterium and to be synthesized by the bacterium in response to the presence of PCP has been purified. This new protein, which is herein named PcpA, has a molecular weight of approximately 30,000. PcpA is believed to be involved in PCP breakdown either at the stage of PCP uptake into the cell or as a component of a PCP-degrading enzyme complex.

Two other proteins are identified, characterized and provided in purified form for the first time by this invention. These two proteins, herein named PcpB and PcpC, are shown to be enzymes which catalyze successive steps in the PCP breakdown pathway of the Flavobacterium. PcpB (also termed pentachlorophenol hydroxylase by the inventors) catalyzes the conversion of PCP to 2,3,5,6-tetrachloro-p-hydroquinone (TeCH) in the presence of NADPH and oxygen. Purified PcpB is shown to have a molecular weight of approximately 59,000-63,000.

In the presence of the reduced form of glutathione, PcpC (also termed tetrachlorohydroquinone reductase by the inventors) catalyzes the conversion of TeCH to 2,3,6-trichloro-p-hydroquinone (TrCH) and the conversion of TrCH to 2,6-dichloro-p-hydroquinone (DiCH). Purified PcpC is shown to have a molecular weight of approximately 26,000-29,000.

In combination, these proteins therefore catalyze the initial steps of PCP breakdown by this microorganism.

Purified PcpB is further shown to exhibit a wide substrate diversity, including halogen, nitro, amino and cyano substituted phenols. PcpB is also shown to degrade 3,5-dibromo-4-hydroxybenzonitrile (also known as bromoxynil, a widely used herbicide) to produce 2,6-dibromo-p-hydroquinone.

Also provided by the present invention are N-terminal amino acid sequences of these proteins. The purified PcpB protein comprises the amino acid sequence: Ser-Thr-Tyr-Pro-Ile-Asn-Ala-Pro-Gly-Gln-Ser-Ala-Asp-Ala-Ala-Val-Leu-Ile-Val-Gly. The purified PcpC protein comprises the amino acid sequence: Pro-Glu-Val-Ser-Leu-Tyr-Asn-Tyr-Thr-Met-Ser-Ile-Cys-Ser-Met-Lys-Thr-Arg-Leu-Ala. These amino acid sequences from proteins pcpB and pcpC are encompassed in SEQ. ID Nos. 5 and 6, respectively, in the accompanying sequence listing. Based on these amino acid sequences, degenerate oligonucleotides are synthesized corresponding to the DNA sequences encoding these amino acid sequences. The use of these oligonucleotides as hybridization probes to isolate DNA molecules from the total DNA of the Flavobacterium is described.

Purified DNA molecules which hybridize to these oligonucleotide probes are also included within the present invention. Specifically, DNA molecules are provided that include the genes encoding PcpA, PcpB and PcpC. A cloned 2.4 kb EcoRI-AccI DNA molecule (that is, a DNA molecule of size 2.4 kb which is defined by recognition sites for the EcoRI restriction enzyme at one end and the AccI restriction enzyme at the other) which encodes PcpA and hybridizes to a 68 bp oligonucleotide derived from the N-terminal amino acid sequence of PcpA is provided herein. Also provided is a cloned 3.0 kb EcoRI fragment of DNA which encodes PcpB and hybridizes to a probe LX-6 derived from the N-terminal amino acid sequence of PcpB. Similarly, a cloned 4.0 kb EcoRI-PstI DNA molecule which encodes PcpC and hybridizes to a probe LX-7 derived from the N-terminal amino acid sequence of PcpC is also provided herein.

Recombinant DNA vectors containing one or more of these DNA fragments are aspects of this invention, as are microorganisms transformed with these vectors. Nucleotide sequences of the genes encoding PcpA, PcpB and PcpC are also included in this invention. A DNA molecule comprising nucleotide bases 328 to 1944 shown in SEQ ID No. 2 which encode the PcpB protein and a DNA molecule comprising nucleotide bases 388 to 1140 shown in SEQ ID No. 3 which encodes the PcpC protein are part of this invention. In addition, the complete amino acid sequences of the PcpA, PcpB and PcpC proteins, derived by theoretical translation of the corresponding DNA sequences, are also provided in this invention; these amino acid sequences are presented in SEQ ID Nos. 4, 5 and 6, respectively.

Having herein provided DNA molecules which encode PcpA, PcpB and PcpC, correspondingly provided are DNA molecules which hybridize under stringent conditions to one or more of these DNA molecules. Such molecules include DNA molecules differing only by minor sequence changes including nucleotide substitutions, deletions and additions. Also comprehended by this invention are DNA molecules which are fragments of the disclosed cloned DNA molecules, such as oligonucleotides which may be employed as effective DNA hybridization probes or primers useful in the polymerase chain reaction.

Having provided purified PcpA, PcpB and PcpC and cloned DNA molecules which encode these proteins, the present invention also provides for the use of these proteins and DNA molecules in aspects of the bioremediation and dechlorination of PCP containing matter. Improved bioremediation methods comprehended by the current invention include the use of genetically engineered bacteria designed for optimal PCP breakdown characteristics, either in situ or in bioreactors. In this respect, microorganisms transformed with the recombinant DNA vectors will be useful in such bioremediation processes. Encompassed by this invention is a method of dechlorinating PCP comprising exposing material containing PCP to microorganisms transformed with DNA which encodes the PcpB protein. Also encompassed by this invention are transformed microorganisms containing a DNA molecule which encodes the PcpB protein. Additionally, this invention also includes transformed microorganisms containing a DNA molecule which encodes the PcpC protein. Transformed microorganisms containing DNA molecules which encode both of the proteins PcpB and PcpC, as well as transformed microorganisms containing DNA molecules which encode the three proteins PcpA, PcpB and PcpC, are also part of this invention.

Another application comprehended by this invention is the use of the purified proteins (either extracted from the Flavobacterium or from the transformed microorganisms expressing the selected enzyme) to remove PCP from contaminated materials. The present invention also includes methods of using purified proteins PcpB and PcpC for dechlorination of PCP and, in the case of PcpB, for degradation of a wide range of other substituted aromatics. The present invention includes a method of dechlorinating PCP comprising exposing material containing PCP to purified PcpB and also comprehends a method of dechlorinating PCP when the method of dechlorinating further comprises exposing the material to purified PcpC.

Monoclonal antibodies raised against each of three purified proteins are also provided by this invention. These monoclonal antibodies may be used to evaluate the production of the three enzymes in novel isolates of PCP-degrading bacteria and also to determine optimal conditions for PCP breakdown by such bacteria.

The present invention also includes an assay that uses monoclonal antibodies raised against PcpB or, more preferably PcpA, to quantitate the level of PCP contamination at environmental locations. Quantitation of PcP contamination is indirectly measured by quantitating the amount of PcpA or PcpB produced in response to the presence of PCP in a given sample by the Flavobacterium cells which are added to that sample.

DNA sequences derived from the provided DNA molecules or parts of these molecules can be employed in a sensitive assay utilizing the polymerase chain reaction for the presence of PCP-degrading microorganisms at contaminated environmental sites. The DNA sequences of the cloned genes may also be used as disclosed in conjunction with molecular cloning techniques to clone homologous genes from other microorganisms.

The purified enzymes and the genes encoding these enzymes which are disclosed in this invention are novel in the field of microbial PCP breakdown. Those skilled in the art will appreciate the wide potential utility of this invention, which is not limited to the specific experimental modes and materials described herein.

The foregoing and other features and advantages of the invention will become more apparent from the following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1(A) is a graph showing the time course of PCP degradation by Flavobacterium sp. Strain ATCC 39723.

FIG. 1(B) is an SDS-PAGE gel showing PcpA production following induction with PCP.

FIG. 2 is an SDS-PAGE gel of the purified periplasmic protein PcpA.

FIG. 3 is a graphic representation of the results of a Mono Q chromatography column run with a step-and-linear gradient of NaCl ( - - - ) used for the purification of PcpB.

FIG. 4 is a graph showing the results of chromatography on a Superose 6 column with 100 mM NaCl utilized during the purification of PcpB.

FIG. 5 is an SDS-PAGE gel of purified PcpB.

FIG. 6 is a graphic representation illustrating the complete conversion of PCP to TeCH by purified PcpB as measured by HPLC of the reaction mixture.

FIG. 7 is an absorbance spectrum of PcpB.

FIG. 8 is a graph showing the effect of pH on PcpB activity.

FIG. 9 shows structural formulas giving proposed reaction equations for representative substrates of PcpB.

FIG. 10 is a graph showing the results of a Mono Q chromatography column utilized during purification of PcpC.

FIG. 11 is a graph showing the results of chromatography on an FPLC gel filtration column utilized during purification of PcpC.

FIG. 12 is an SDS-PAGE gel of purified PcpC.

FIG. 13 is the N-terminal amino acid sequences of the purified proteins PcpA, PcpB and PcpC as determined by protein sequencing of the purified proteins and theoretical translation of the nucleotide sequences of the corresponding genes pcpA, pcpB and pcpC.

FIG. 14 shows the construction of pCL3, a plasmid vector which expresses the PcpB protein.

FIG. 15, Panel A, shows an SDS-PAGE gel of crude lysates of E. coli JM105 transformed with pCL3.

FIG. 15, Panel B, shows an immunoblot of the SDS-PAGE gel in Panel A. The PcpB protein was detected using polyclonal antibody raised against purified PcpB.

FIG. 16 shows HPLC chromatograms demonstrating the conversion of PCP to TeCH by cell extracts of E. coli JM105 transformed with pCL3.

FIG. 17 shows HPLC chromatograms demonstrating the conversion of PCP to TeCH by intact E. coli JM105 cells transformed with pCL3 and induced with IPTG.

Sequence Listings

Nucleotide and amino acid sequences are set forth in the "Sequence Listing" appended hereto. Sequence ID No. 1 sets forth the nucleotide sequence of the pcpA gene and the amino acid sequence of the protein encoded by this gene. Sequence ID No. 2 sets forth the nucleotide sequence of the pcpB gene and the amino acid sequence of the protein encoded by this gene. Sequence ID No. 3 sets forth the nucleotide sequence of the pcpC gene and the amino acid sequence of the protein encoded by this gene. Sequence ID Nos. 4, 5 and 6 set forth the amino acid sequences of the proteins PcpA, PcpB and PcpC, respectively. Sequence ID Nos. 7, 8, 9 and 11 set forth the nucleotide sequences of primers LX-1B, LX-2B, LX-6 and LX-7, respectively. Sequence ID No. 10 sets forth the N-terminal amino acid sequence of PcpC as determined by protein sequencing.

DETAILED DESCRIPTION

According to the present invention, purified preparations of three bacterial proteins, PcpA, PcpB and PcpC, are provided. As used herein and in reference to the preparations of purified PcpA, PcpB and PcpC, the term "purified" refers to a preparation of a protein (either PcpA, PcpB or PcpC) that is purer than naturally occurring in nature. More specifically, a purified protein is purified to the extent that other proteins represent no more than 50% of the final protein preparation, and more preferably no more than 10% of the final protein preparation. In particularly preferred embodiments, the proteins are purified to provide protein preparations that are essentially free of biological agents normally associated with PcpA, PcpB or PcpC in their in vivo physiological milieu. Such essentially pure enzymes are free of other proteins when assessed electrophoretically by sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualization by staining with Coomassie brilliant blue R-250. In the most preferred embodiments, the proteins are purified to an extent sufficient to allow the determination of N-terminal amino acid sequences by protein sequencing.

Further, DNA molecules encoding these three proteins have been isolated and cloned. These cloned DNA molecules have been incorporated into vectors suitable for the transformation of bacterial cells, each vector carrying a gene which encodes one of the three proteins PcpA, PcpB or PcpC. The three genes which encode the PcpA, PcpB and PcPC proteins are herein named pcpA, pcpB and pcpC, respectively. The DNA molecules, incorporated into recombinant DNA vectors, have been transformed into microorganism host cells.

Materials and methods used are presented in the following section and with reference to the Figures herein provided. The materials and methods are presented by way of illustration and represent particular embodiments of the invention. Those skilled in the art will recognize that these embodiments can be modified in arrangement and detail without departing from the principles of the present invention.

Bacterial Strains and Sources of Chemicals

Flavobacterium sp. Strain ATCC 39723 was obtained from Dr. R. Crawford (University of Idaho) and is available from the American Type Culture Collection as Accession Number 39723. This microorganism is the subject of U.S. Pat. No. 4,713,340 to Crawford, herein incorporated by reference. The standard laboratory strains Escherichia coli HB101 and JM105 were used as recombinant hosts in cloning experiments.

Flavobacterium strains were cultured in mineral medium with 4 g/l sodium glutamate as the growth substrate, and PCP degradation was induced by adding PCP to a final concentration of 40 μg/ml to cultures at early log phase (A₆₀₀ <1.0) (Xun and Orser, 1991a). The induction time required for PCP degradation was usually 1 to 2 hrs. E. coli strains were routinely grown at 37° C. in Luria broth or on Luria agar (Sambrook et al., 1989). The antibiotics tetracycline and ampicillin were used at 15 and 50 μg/ml, respectively. Both antibiotics were purchased from Sigma Chemical Co. (St. Louis, Mo.).

Unless otherwise stated, reagents and enzymes were obtained from the suppliers listed below. Restriction endonucleases, RNA standards, and protein molecular weight standards were purchased from Bethesda Research Laboratories, Inc. (Gaithersburg, Md.), and used as recommended by the supplier. T4 DNA ligase, DNA polymerase I, avian myeloblastosis virus reverse transcriptase, mung bean exonuclease, exonuclease III, random-primed DNA labeling kit, and sequencing reagents were purchased from United States Biochemical Corp., Cleveland, Ohio. X-ray film was purchased from Kodak Co., Rochester, N.Y.; [α-³² P]dCTP, [gamma-³⁵ S]dATP, and [α-³⁵ S]dATP were from New England Nuclear Corp., Boston, Mass.; DNA Taq polymerase was purchased from Perkin-Elmer Corp. (Norwalk, Conn.). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). Standard molecular biology laboratory techniques were performed according to Sambrook et al. (1989) unless stated otherwise.

Identification and Purification of PcpA

The identification and purification of the pentachlorophenol-induced periplasmic protein PcpA are described in Xun and Orser (1991a), herein incorporated by reference.

Identification of PcpA

Subcellular fractions were prepared from Flavobacterium sp. Strain ATCC 39723, as described below, in order to localize PCP degrading activity. PCP degrading activity was assayed by the Leuco crystal violet assay as described below and in Xun and Orser (1991b), herein incorporated by reference. It was found that when cells were broken by sonication or French press that PCP degrading activities ceased and furthermore, that no PCP degrading activity was detectable in cytoplasmic, inner or outer membrane fractions. PCP degrading activity was undetectable in membrane vesicles isolated from the Flavobacterium. It was subsequently determined that the presence of EDTA in the cell suspension buffer was responsible for the arrest of PCP degrading activity. Removal of EDTA by centrifugation and resuspension of the cells in EDTA-free buffer did not restore enzyme activity, leading to the conclusion that EDTA released PCP degrading proteins from the bacterial cell wall.

An additional protein band of approximately 30 kDa. was detected in PCP-induced cells when proteins released by EDTA were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). This 30 kDa. protein was found to be required for PCP degradation. Following the addition of PCP to cell medium, this protein was observed to be induced and to reach a maximum intensity as visualized by SDS-PAGE after about one hour and to be essentially absent after six hours. PCP degradation started at 40 min. after the addition of PCP, and the rate reached its maximum at one hour. After six hours, the protein was no longer visible by SDS-PAGE, and the PCP degrading activity could no longer be measured in a culture. These results are shown in FIG. 1. FIG. 1(A) diagrams PCP dechlorination as measured spectrophotometrically following PCP induction at time zero; FIG. 1(B) shows a time course of periplasmic proteins released by EDTA following PCP induction at time zero, monitored by SDS-PAGE. Sizes are indicated in kilodaltons to the left of the gel photograph. The identified 30 kDa. protein was designated PcpA.

PcpA Is Absent from 2PCP Mutants

Two PCP mutants, F2 and F23 (Steiert and Crawford, 1986), which do not degrade PCP, did not produce the periplasmic protein PcpA following treatment with PCP. Otherwise, the mutants were identical to the wild type Flavobacterium sp. Strain ATCC 39723 in their growth and periplasmic protein patterns as visualized by SDS-PAGE.

Purification of PcpA

The 30 kDa. periplasmic protein PcpA was purified from Flavobacterium sp. Strain ATCC 39723 by five successive purification steps: Periplasmic protein extraction, streptomycin sulfate precipitation, ammonium sulfate fractionation, phenyl-agarose hydrophobic chromatography, and DEAE-Sepharose ion exchange chromatography. SDS-PAGE of the purified PcpA protein is shown in FIG. 2. Lane 1 of FIG. 2 shows the purified protein preparation and lane 2 shows the proteins of the periplasmic fraction from PCP-induced cells of Flavobacterium sp. Strain ATCC 39723.

Method of PcpA Purification

(i) Periplasmic Protein Extraction. One liter of PCP-induced Flavobacterium cells was harvested by centrifugation at 7,000×g for 10 min. The cells were resuspended in 100 ml of 10 mM EDTA-25 mM Tris (pH 8.0). The cell suspension was kept on ice for one hour and then centrifuged at 7,000×g for 10 min. The supernatant containing the periplasmic proteins was saved.

(ii) Streptomycin Treatment. Solid streptomycin sulfate was added to the supernatant to a final concentration of 0.5% As soon as the streptomycin dissolved, the suspension was centrifuged at 10,000×g for 10 min. and the pellet was discarded.

(iii) Ammonium Sulfate Precipitation. Solid ammonium sulfate was added to the protein solution to 65% saturation. When the ammonium sulfate was completely dissolved, the suspension was left at room temperature for 30 min. The protein precipitate was collected by centrifugation at 10,000×g for 10 min.

(iv) Phenyl-Agarose Chromatography. A column (30 by 1.5 cm) containing 40 ml (bed volume) of phenyl-agarose (Sigma) packed with 20 mM Tris and 2.5 mM EDTA (pH 8.0) in a 6.2° C. cold room was equilibrated with the same buffer containing 20% ammonium sulfate at a flow rate of 30 ml/h via a peristaltic pump (Pharmacia Co., Piscataway, N.J.). The ammonium sulfate precipitate from a 4-liter culture was resuspended in 20 ml of 20 mM Tris-2.5 mM EDTA (pH 8.0) and adjusted to 20% ammonium sulfate by adding solid ammonium sulfate. The solution was centrifuged, the supernatant was loaded onto the phenyl-agarose column, and the protein was eluted with a gradient of 20 to 0% ammonium sulfate in 500 ml of the same buffer. The protein eluted between 7 and 1% ammonium sulfate. Fractions (8 ml) were collected with a microfractionator (Bio-Rad, Richmond, Calif.), and those containing the protein as detected by SDS-PAGE were pooled and concentrated to a volume of 10 ml by dialysis against polyethylene glycol (Sigma) for four hours, using Spectrapor membrane tubing (Spectrum Medical Industries, Inc., Los Angeles, Calif.) with a molecular weight cutoff of 12,000 to 14,000. The sample was further dialyzed against 20 mM Tris (pH 8.0) overnight.

(v) DEAE-Sepharose Chromatography. A column (30 by 1 cm) containing 40 ml of DEAE-Sepharose packed with 20 mM Tris (pH 8.0) in a 6.2° C. cold room was equilibrated with 80 ml of the Tris buffer at a flow rate of 30 ml/h via a peristaltic pump. The 20 mM Tris buffer-dialyzed sample was loaded onto the column and washed with 40 ml of the Tris buffer. The protein was eluted by a linear gradient of 0.1 to 0.4M NaCl in 500 ml of 20 mM Tris (pH 8.0). The protein eluted between 0.24 to 0.29M NaCl. The fractions containing the protein were collected and concentrated to 1 ml by dialysis against polyethylene glycol. The sample was stored at -20° C.

Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE)

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by the method of Laemmli (1970). Nondenaturing PAGE was done in the same way but without the addition of SDS, and the acrylamide gradient was formed with a gradient maker (Ann Arbor Plastics, Ann Arbor, Mich.). Gels were stained for protein with Coomassie brilliant blue R-250. Protein quantification was performed using the Bradford protein assay (Bradford, 1976).

Identification and Purification of PcpB

The identification and purification of PcpB are described in Xun and Orser (1991c), herein incorporated by reference.

Identification of PcpB

The first step of PCP degradation by microorganisms including Flavobacterium sp. Strain ATCC 39723 is believed to be a hydrolytic dechlorination producing 2,3,5,6-tetrachloro-p-hydroquinone (TeCH). This strain also degrades triiodophenol (TIP) to produce iodide, which reaction can be measured by the Leuco crystal violet assay. It was concluded that the degradation of PCP to TeCH and the breakdown of TIP to yield iodide proceed by the same mechanism, and that a single hydrolytic enzyme is responsible for both these reactions. A scheme for purifying this enzyme, named pentachlorophenol hydroxylase or PcpB, was therefore devised using the Leuco crystal violet assay as described below to detect the presence of the enzyme.

Purification of PcpB

Purification of PcpB was facilitated by the ability of the enzyme to release iodide from triiodophenol. This activity, as measured by the Leuco crystal violet assay was utilized to detect the presence of PcpB throughout the enzyme purification scheme which is described below. Standard techniques were as described for PcpA purification unless otherwise stipulated.

Method of PcpB Purification

All operations were performed at 4° C., and the buffer was 20 mM Tris hydrochloride (pH 8.0) unless mentioned otherwise. When ammonium sulfate was added, the buffer also contained 1 mM EDTA. Ammonium sulfate saturation levels referred to a temperature of 25° C.

(i) Extraction of Cells. Flavobacterium sp. Strain ATCC 39723 was grown as described above. Frozen (-20° C.) cell paste was thawed in water and suspended in buffer at a concentration of approximately 0.2 g of cells per ml. The protease inhibitor phenylmethylsulfonyl fluoride was freshly prepared in absolute ethanol at a concentration of 250 mM and added to the cell suspension at a final concentration of 5 mM. The slurry was passed through an Aminco French pressure cell (model 4-3398) twice at 12,000 lb/in². The product was centrifuged at 17,000×g for 10 min., and the supernatant was saved. The precipitate was resuspended in the buffer, passed through a French pressure cell twice, and centrifuged as described above. The supernatant was combined with the previous supernatant, and the precipitate was discarded.

(ii) Protamine Sulfate Fractionation. A 2% solution of protamine sulfate in 20 mM Tris buffer (pH 8.0) was added to the supernatant slowly with constant stirring to a final concentration of 0.1 mg/ml. After 5 min. of stirring, the mixture was centrifuged at 17,000×g for 10 min., and the precipitate was discarded.

(iii) Ammonium Sulfate Fractionation. Solid ammonium sulfate was added to the supernatant to 40% saturation with constant stirring. The pH of the solution was not adjusted. After 10 min. of stirring, the mixture was centrifuged (17,000×g for 10 min.), and the precipitate was discarded. Additional solid ammonium sulfate was added to 60% saturation with constant stirring. After 10 min. of stirring, the mixture was centrifuged (17,000×g for 10 min.). The precipitate was saved, and the supernatant was discarded.

(iv) Phenyl Agarose Chromatography. The precipitate was suspended with an equal volume of buffer. The suspension was centrifuged (17,000×g for 10 min.), and the precipitate was discarded. The supernatant was loaded onto a phenyl agarose (Sigma) column (30 by 1.5 cm) previously equilibrated with buffer containing 10% ammonium sulfate. The enzyme was eluted with 300 ml of a 10-to-0% ammonium sulfate gradient. The enzyme eluted to around 5% ammonium sulfate. The fractions containing enzyme activity were pooled and precipitated by adding ammonium sulfate to 70% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.).

(v) Mono Q Chromatography. The proteins were resuspended in 3 ml of 20 mM Tris hydrochloride buffer (pH 8) and dialyzed against 1 liter of the same buffer for 1 hour. The protein solution was injected onto a Mono Q HR 5/5 column (Pharmacia) equilibrated with 20 mM Tris buffer. Proteins were eluted with a step-and-linear gradient of sodium chloride (percentage of 1M NaCl in volume of 20 mM Tris: 0%, 2 ml; 35%, 4 ml; 35 to 55%, 9 ml linear gradient; 100%, 4 ml; 0%, 2 ml) by a Pharmacia fast protein liquid chromatography system (FPLC) (Pharmacia LKB Programmer GP-250 Plus and P-500 pump). Fractions of 1 ml were collected. A single PcpB activity peak was associated with a single protein peak around an NaCl concentration of 0.45M. The results of this chromatography are shown in FIG. 3; PcpB activity was associated with the major protein peak at fractions 10 and 11 shown in the figure.

The fractions containing enzyme activity were pooled and precipitated by adding ammonium sulfate to 75% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.).

(vi) Gel Filtration Chromatography. The protein precipitate was dissolved with an equal volume of buffer and injected onto a Superose 6 column (Pharmacia) equilibrated with 20 mM Tris buffer containing 100 mM NaCl. The enzyme was eluted with the same buffer by the Pharmacia FPLC system. Fractions of 1 ml were collected. PcpB activity was associated with a major protein peak with a retention volume of 16 ml. FIG. 4 shows a typical result obtained from this purification stage; PcpB activity was associated with the major protein peak at fractions 16 and 17. The fractions containing the enzymatic activity were pooled and precipitated by adding ammonium sulfate to 75% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.).

(vii) Crystallization. The proteins were dissolved with 0.4 ml of saline (0.9%) KP_(i) buffer (50 mM, pH 7.4) and transferred to a 1.5 ml microcentrifuge tube. Saturated ammonium sulfate solution was added dropwise until a faint, permanent turbidity developed. The sample was then quickly centrifuged (16,000×g for 10 min.) to remove any denatured proteins. Additional ammonium sulfate was added to the supernatant to form microform crystals of the enzyme. The crystal was recovered by centrifugation (16,000×g for 10 min.). The enzyme was stored at -20° C.

Leuco Crystal Violet Assay

The Leuco crystal violet assay was performed as described by Black and Whittle (1967) with the modifications described by Xun and Orser (1991b). All assays were performed in 50 mM Tris-H₂ SO₄ (pH 8.0) with 100 μM TIP.

Solutions:

1) Citric buffer solution, pH 3.8 was prepared as follows: 350 ml 2N NH₄ OH solution were added slowly with mixing to 670 ml of 0.333 n citric acid solution. 80 g of NH_(4H) 2PO₄ were then added to the mixture and stirred to dissolve. The pH was adjusted to 3.8.

2) 0.15% potassium monopersulfate triple salt in ddH₂ O (Fluka Chemical Co., St. Louis, Mo.).

3) Leuco crystal violet indicator was prepared as follows: 3.2 ml of concentrated sulfuric acid was added to 200 ml ddH₂ O in a brown glass container and mixed with a magnetic stir bar at moderate speed. Leuco crystal violet (1.5 g) (Sigma Chemical Co., St. Louis, Mo.) was added along with 800 ml of 0.31% HgCl₂ with mixing until the Leuco crystal violet was dissolved. The pH was adjusted to 1.5 or less with H₂ SO₄.

Free iodide in culture supernatants was measured as follows:

0.5 ml of bacterial culture supernatant was transferred to a 1.5 ml microcentrifuge tube, and 0.1 ml of citric buffer solution (pH 3.8) was added. After mixing, the mixture was centrifuged in a microcentrifuge at 16,000×g for 5 min. A 0.3 ml aliquot of the supernatant was removed to another microcentrifuge tube and diluted to 0.5 ml with ddH₂ O, and thereafter 10 μl of 0.15% potassium monopersulfate triple salt were added. This was then mixed and left to stand for approximately 1 min. 10 μl of Leuco crystal violet indicator were added, and following further mixing, the preparation was diluted to a volume of 1 ml with ddH₂ O. Absorbance was then read spectrophotometrically at 592 nm. Iodide standards were prepared in the same buffer solution as in the assay.

Free iodide in enzyme reaction mixtures was measured as follows:

A 0.25 ml sample was placed in a 1.5 ml microcentrifuge tube, 0.05 ml of citric acid buffer were added, and the resulting mixture boiled for 3 min. Thereafter, the tube was centrifuged at 15,000×g for 2 min. 0.25 ml of supernatant were transferred to another 1.5 ml microcentrifuge tube, and 0.25 ml of ddH₂ O and 10 μl of potassium peroxymonosulfate solution were added. The tube contents were mixed and allowed to stand for 1 min. prior to the addition of 10 μl of crystal violet solution. After this addition, the solutions were further mixed and diluted to 1 ml with ddH₂ O. Absorbance was measured at 592 nm. The iodide standards were prepared in the same reaction mixture without triiodophenol.

PCP Degradation as an Assay of PcpB Activity

PcpB activity was assayed by measuring the depletion of PCP and/or the production of TECH. The standard assay system contained 100 mM potassium phosphate (KP_(i)) or Tris acetate buffer (pH 7.5), 100 μM NADPH solution, 100 μM PCP, 0.5% Tween 20, 10 μM MgSO₄, and enzyme in a 50 μl volume at a constant temperature of 25° C. Tween 20 and MgSO₄ were found to increase the dehalogenating activity in the cell extract, and therefore they were included in the standard assay used in enzyme purification. MgSO₄ was later determined not to enhance the activity of the purified enzyme. Tween 20 was determined to facilitate reproducible quantitation of PCP by high-performance liquid chromatography (HPLC) analysis as described below. A 10 μl volume of reaction mixture was injected onto the HPLC column at 4 min. after the start of the reaction. One unit of enzyme activity was defined as the amount of enzyme that catalyzed the production of 1 μmol of TeCH or the consumption of 1 μmol of PCP per min under the assay conditions. The reaction rate was linear for about 5 min. with 0.05 to 0.04 U of the enzyme, and then the reaction rate decreased gradually over the next 15 min.

Analytical Methods for PcpB Purification

PCP and TeCH were analyzed by HPLC (Waters, Milford, Mass.) on a Nova-Pak C18 column (3.9 by 150 mm) with an 11 mM H₃ PO₄ -acetonitrile gradient (acetonitrile concentrations 10 to 65% [5 min.] and 65% [10 min.]). Maximum absorption at 310, 320, and 330 nm was recorded by use of a Waters 490E programmable multiwavelength UV detector connected to a Maxima NEC computer workstation (Waters). The retention times of PCP and TeCH were 11.5 and 8.9 min., respectively. PCP and TeCH were quantified by comparing their peak areas with the peak areas of authentic standards. NADPH concentration in the reaction mixture was measured by HPLC on the Nova-Pak C18 column with an isocratic 50 mM KP_(i) buffer (ph 8.0). Flavins were determined by HPLC on a μCarbohydrate analyzing column (3.9 by 300 mm) (Analytical Sciences Incorporated, Santa Clara, Calif.) with an isocratic solution of acetonitrile-distilled water (60:40, vol/vol). Protein purity was checked by HPLC on a Delta Pak C18 30 nm column (3.9 by 150 mm) with a water-acetonitrile gradient containing 0.1% trifluoroacetic acid (acetonitrile concentrations, 15% [1 min.], 15 to 40% [1 min.], 40% [2 min.], 40 to 75% [8 min.], 75 to 100% [ 1 min.], 100% [7 min.], 100 to 15% [1 min.]) at a flow rate of 1 ml/min.

Results

The results of a typical PcpB purification are summarized in Table 1.

                                      TABLE 1                                      __________________________________________________________________________     Purification of PcPB                                                                                    Activity.sup.a                                                                          Recovery                                     Purification step                                                                           Vol (ml)                                                                            Protein (mg)                                                                          Total                                                                              Specific                                                                            (%)                                          __________________________________________________________________________     Crude extract                                                                               72.00                                                                               1,849  133.1                                                                              0.072                                                                               100                                          Protamine sulfate                                                                           74.00                                                                               1,398  132.8                                                                              0.095                                                                               100                                          fractionation                                                                  Ammonium sulfate frac-                                                                      18.00                                                                                 460  127.4                                                                              0.277                                                                               95.7                                         tionation (40 to 60%)                                                          Phenyl agarose                                                                              5.50   62   117.9                                                                              1.89 88.6                                         chromatography                                                                 Mono Q chromatography                                                                       0.72    9   89.7                                                                               9.75 67.4                                         Superose 6   0.55    6   62.5                                                                               11.00                                                                               47.0                                         chromatography                                                                 crystallization                                                                             0.61    5   55.4                                                                               11.54                                                                               41.6                                         __________________________________________________________________________      .sup.a Activity was estimated by TecH production in 100 mM KP.sub.i buffe      (pH 7.5). TecH standards were prepared in the mixture without PCP and          incubated at room temperature for 4 min. before being analyzed by HPLC.  

Since the enzyme also converts PCP to TeCH, the enzymatic activities in Table 1 were determined from TeCH production, as described above. In the reaction mixture containing crude cell extract, only a very small quantity of TeCH was detected by HPLC, about 1 to 2 μM within 4 min., which gave a distinct peak by HPLC analysis. There was no TeCH detectable after 15 min. of incubation with crude extracts. The purification scheme, consisting of seven steps, resulted in a 160-fold purification of PcpB relative to the crude cell extract (the specific activity increased from 0.072 to 11.51 U/mg of protein). In preferred embodiments, purified PcpB has a specific activity greater than 1 U/mg of protein, and in a most preferred embodiment, purified PcbB has a specific activity greater than 10 U/mg protein. Approximately 41.6% of the activity of PcpB in the crude extract was recovered. A typical elution profile of the proteins chromatographed on an FPLC Mono Q column (Pharmacia) is shown in FIG. 3. The activity of PcpB was found only in a large protein peak located in fractions 10 and 11, a 2 ml volume. The peak was not present when proteins from PCP-uninduced cells were chromatographed, indicating that the enzyme is PCP induced. After Mono Q chromatography, the proteins were chromatographed on a Superose 6 column (Pharmacia), and a typical chromatograph is shown in FIG. 4. After crystallization, PcpB was a single band by SDS-PAGE analysis; FIG. 5 shows a typical gel with molecular size markers (in kilodaltons) in lane 1 and 2.5 μg of PcpB in lane 2. Also, after crystallization, PcpB was detected as a major peak which represented 95% of the total peak area on reverse-phase HPLC column chromatography.

After the initial purification of PcpB by using TIP as the substrate, PCP was tested as the substrate. PCP and TeCH were quantified by HPLC as described below. PCP was subsequently used in place of TIP for routine enzyme purification. The enzyme completely converted 37 μM PCP to TeCH in 100 mM KP_(i) buffer (pH 7.0) within 20 min. in the presence of 100 μM NADPH (FIG. 6). The reaction was confirmed to be enzymatic because controls without enzyme or with boiled enzymes exhibited no change in PCP concentration after one hour of incubation. The TeCH produced displayed the same retention time on HPLC as authentic TECH.

Properties of PcpB

The molecular weight of PcpB was estimated to be about 63,000 (FIG. 5). A major band with a molecular weight of 63,000 and a minor band of molecular weight 132,000 were detected on a native gradient PAGE, indicating that it exists mainly as a monomer but also can associate as dimers under native electrophoresis conditions. For gel filtration, when the elution buffer contained 100 mM NaCl, the protein eluted as a single peak on a Superose 6 column (Pharmacia) with a retention volume of 16.0 ml, which was the same as the retention volume of bovine serum albumin (16.0 ml). When NaCl was not used in the elution buffer, the protein eluted as a single peak with a retention volume of 12.8 ml. The data from gel filtration chromatography indicated that the protein was a monomer with a molecular weight of 66,000 when NaCl was used in the elution buffer and that the protein was a multimer with a molecular weight higher than 300 kDa. when NaCl was not used in the elution buffer. Since NaCl was not included in the reaction mixture, the enzyme existed as a multimer and therefore the multimer is functional. The discrepancy between the molecular weights of PcpB determined by SDS-PAGE and gel filtration is likely accounted for by differences between the two techniques and inherent experimental variation. Multimeric forms of PcpB, defined as a protein polymer with repeating subunits of PcpB and having the activity of converting PCP to TeCH, are therefore comprehended by this invention. The isoelectric point (pI) of the purified protein was determined to be approximately 4.3 by isoelectric focusing (IEF).

The enzyme was a flavoprotein exhibiting a typical flavoprotein spectrum, with a peak at 446 nm. FIG. 7 shows a typical absorbance spectrum for PcpB determined with a protein solution contained 24.2 μM PcpB in 20 mM Tris hydrochloride (pH 8.0). The addition of NADPH to 150 μM did not reduce the flavoprotein absorption at 446 nm. A solution containing 24.2 μM protein (1.5 mg of protein per ml; molecular weight, 63,000) gave an A₄₄₆ of 0.175. The flavin content was calculated to be 15.5 μM by using a molar extinction coefficient of 11,300 for flavin adenine dinucleotide (FAD) (Strickland and Massey, 1973). The molar ratio of flavin to protein was 0.64. The result indicated that each PcpB molecule contained one flavin molecule. The reduced amount of flavin present in the purified protein could be due to the loss of the flavin during purification. The loss of flavin during purification of other flavin monooxygenases has been reported (Flashner and Massey, 1974; Strickland and Massey, 1973). The flavin in the protein was easily extracted by trichloroacetic acid (Mayhew and Massey, 1969) and was identified as FAD on the basis of the retention time by HPLC (retention times, 5.5 min. [flavin mononucleotide, FMN] 7.1 min. [FAD], and 7.1 min. [sample]).

The enzyme was very stable as both an ammonium sulfate precipitate and in solution at a concentration greater than 1 mg/ml at -20° C. There was no measurable loss of activity over a period of 2 months. At 4° C., about 50% of the activity was gone after 1 week. Enzymatic activity in the cell extract and in the 40 to 60% ammonium sulfate precipitation fraction was completely lost after 3 days at 4° C.

Optimal Conditions for Enzymatic Activity of PcpB

Results from studies of PcpB activity at different pH values are shown in FIG. 8. In FIG. 8, points marked O represent activity assayed in 100 mM KP_(i) buffer, points marked * represent activity assayed in 100 mM Tris buffer.

The optimal pH for PcpB activity was between 7.5 and 9.0. However, under the optimal pH conditions, the enzymatic product, TeCH, was highly unstable. TeCH oxidized and polymerized to a more hydrophilic compound which was eluted from the reverse-phase HPLC column in the void volume. At a pH lower than 7.5, TeCH was relatively stable. At pHs 7.0, 6.5, and 6.0, the enzyme activity levels were about 90, 57, and 28%, respectively, of that at pH 7.5. The optimal temperature of the enzymatic reaction was between 35° and 40° C. Greater than 75% of the maximal activity was retained at 45° C. The enzymatic activity was completely absent at 50° C. For the purified enzyme, MgSO₄ was not required for activity because the addition of MgSO₄ or EDTA to 5 mM did not influence the enzymatic activity. This indicates that the enzyme does not require divalent cations for enzymatic activity. Non-ionizing detergents were not required for enzymatic activity for the purified enzyme, as measured by TeCH production. However, Tween 20 was left in the reaction assay because it was found to contribute to the reproducibility of results in the quantitation of PCP by HPLC analysis.

Methods for Determination of pH and Temperature Optima for PcpB

PcpB activity was measured at various pH values within the rage of 6.0 to 9.0 by using 100 mM KP_(i) buffer (pH 60 to 8.0) or 100 mM Tris acetate buffer (pH 7.5 to 9.0) in a total volume of 50 μl . The enzyme reaction mixture was otherwise the same as defined above for the enzyme assay. The temperature optimum for the enzyme activity was determined in a similar way in KP_(i) buffer (pH 7.5). The reaction mixture without NADPH was incubated at the corresponding temperature for 4 min., and then 0.5 μl of 10 mM NADPH solution was added to the reaction mixture to start the reaction.

Determination of the Isoelectric Point of PcpB

The isoelectric point (pI) of PcpB was determined using analytical isoelectric focusing (IEF) gels which comprised 5% acrylamide and contained commercially available ampholytes (LKB, Bromma, Sweden) (pH 3.5 to 10). IEF gels were run on an LKB 2117 Multiphor II electrophoresis system as directed by the manufacturer (LKB). A 10 μg amount of purified protein was applied to the IEF gel, and the pI was estimated from the IEF markers (Sigma).

PcpB Exhibits a Diverse Substrate Range

The ability of Flavobacterium sp. Strain ATCC 39723 to degrade chlorinated phenols, triiodophenol, and tribromophenol has previously been reported (Steiert et al., 1987; Xun and Orser, 1991b). The work reported by Xun et al. (1992a) herein incorporated by reference and described below, indicates that PcpB catalyzes the primary metabolic attack on these compounds. PcpB displaces an unusually diverse range of substituents from the para position of polyhalogenated phenols. In addition to removal of halogen, PcpB removes nitro, amino, and cyanol groups from a benzene ring. PcpB also converts 3,5-dibromo-4-hydroxybenzonitrile (also known as bromoxynil, a widely used herbicide) to 2,6-dibromo-p-hydroquinone. The substrates for PcpB reported here are just those compounds tested to date; the enzyme may use additional substrates not tested. This work demonstrates that PcpB utilizes a wide substrate range, and that the gene which encodes this enzyme would be appropriate for use in constructing many microorganisms with novel biodegradative phenotypes using recombinant DNA techniques.

Methods for Determining PcpB Substrates

Stock Solutions

A stock solution of PCP (concentration, 37.6 mM) was prepared in 0.2M NaOH and stored in the dark at room temperature. Stock solutions of all other halogenated aromatic compounds (concentration, 20 mM) were prepared in absolute ethanol and stored at 4° C. in the dark. The NADPH solution of 10 mM was freshly prepared in 1 mM dithiothreitol and 1 mM Tris base (pH 13).

Enzyme Reaction Conditions

PcpB was purified as described above. Enzyme reactions were conducted in 80 mM potassium phosphate buffer (pH 7.5) with 0.5% Tween 20 at 23° C. The enzyme concentration was 500 μg/ml in all reactions.

Analytical Methods for Determining PCpB Substrate Range

All of the aromatic substrates and the corresponding enzyme product (aromatic portion) were monitored by reverse phase high-performance liquid chromatography (HPLC) analysis as described above for PCP and TeCH analysis, except that the absorptions at 285, 300, and 310 nm were recorded by using a Waters 490 E programmable multiwavelength UV detector. Gas chromatography/mass spectrometry (GC/MS) analysis was done as previously described (Topp and Akhtar, 1990).

I was measured by the Leuco crystal violet assay described above. NO₂ was colorimetrically measured using the N-(1-naphthyl) ethylenediamine dihydrochloridesulfanilamide reagent method (American Public Health Association, 17th Ed., 1989). NH₃ was colorimetrically measured by Nesslerization method (American Public Health Association, 17th Ed., 1989). CN was measured by the colorimetric method of Nagashima (1984). NH₂ OH was measured by the method of Magee and Burris (1954). All the assays were proportionally reduced to 1 ml assay volume, and standards of all the tested chemicals were prepared in the enzyme assay mixture without the enzyme.

Results

Table 2 summarizes the chemicals tested as substrates for PcpB activity and the end product detected as a result of PcpB breakdown of these chemicals.

                  TABLE 2                                                          ______________________________________                                         Substrate Disappearance in the Presence of PcpB                                                 (%) Relative                                                  Substrates       Activity   End Products.sup.a                                 ______________________________________                                         Pentachlorophenol                                                                               100        Tetrachloro-p-                                                                 hydroquinone                                       Pentafluorophenol                                                                                47        N.I..sup.b                                         2,3,5,6-Tetrachlorophenol                                                                       1212       Tetrachloro-p-                                                                 hydroquinone                                       2,4,6-Triiodophenol                                                                             172        2,6-Diiodo-p-                                                                  hydroquinone                                       2,4,6-Tribromophenol                                                                             35        2,6-Dibromo-p-                                                                 hydroquinone                                       Trichlorophenol                                                                2,4,6-            29        2,6-Dichloro-p-                                                                hydroquinone                                       2,3,6-           302        2,3,6-Trichloro-                                                               p-hydroquinone                                     2,3,4-            29        N.I..sup.b                                         3,4,5.sup.c       0         None                                               2,6-Dibromophenol                                                                               605        2,6-Dibromo-p-                                                                 hydroquinone                                       Dichlorophenol                                                                 2,6              344        2,6-Chloro-p-                                                                  hydroquinone                                       2,3-              24        N.I..sup.b                                         3,5-.sup.c        0         None                                               4-Amino-2,6-dichlorophenol                                                                       93        2,6-Chloro-p-                                                                  hydroquinone                                       2,6-Dibromo-4-nitrophenol                                                                        62        2,6-Dibromo-p-                                                                 hydroquinone                                       3,5-Diiodo-4-    835        2,6-Diiodo-p-                                      hydroxybenzonitrile         hydroquinone                                       3,5-Dibromo-4-   591        2,6-Dibromo-p-                                     hydroxybenzonitrile         hydroquinone.sup.d                                 ______________________________________                                          .sup.a All enzyme reactions were conducted in a reaction mix volume of 10      μl, containing 10 nmoles of substrate, 40 nmoles of NADPH. The              substrate depletion and product production were measured by HPLC or GC/MS      analysis. Relative enzyme activity for PCP was assumed to be 100%; the         relative enzyme activities for other compounds were compared with that fo      PCP.                                                                           .sup.b N.I., not identified; however the formation of a reaction product       was shown by HPLC.                                                             .sup.c Except 3,4,5trichlorophenyl and 3,5dichlorophenol, all the              substrates listed in the table were completely oxidized by the enzyme.         Monochlorophenol and phenol were not degraded by the enzyme.                   .sup.d From Topp et al. (1992).                                          

In all cases examined, PcpB converted the substituted phenol to the corresponding hydroquinone. PCP and 2,3,5,6-tetrachlorophenol were converted to a compound with the same HPLC retention time as that of TeCH (Xun and Orser, 1991c). TeCH was confirmed as the end-product by GC/MS (data not shown). PcpB oxidized 2,4,6-triiodophenol (TIP) to a product with a retention time of 7.75 min. by GC analysis and mass spectrum consistent with that expected for 2,6-diiodo-p-hydroquinone (DIHQ, molecular formula of C₆ H₄ I₂ O₂). The base peak was at M/Z 362 (M+), and major fragments were found at M/Z 235 (loss of I from 362), 207 (loss of CO from 235), and 108 (loss of 2 I from 362). The end product of 3,5-diiodo-4-hydroxylbenzonitrile was also identified as DIHQ because it shared the same retention time with DIHQ produced from TIP by HPLC analysis. Incubation of 2,3,6-trichlorophenol with PcpB yielded a product with a retention time of 4.65 min. by GC analysis and a mass spectrum consistent with that expected for 2,3,6-trichloro-p-hydroquinone (molecular formula of C₆ H₃ Cl₃ O₂). The relative intensities of the peaks at M/Z 212 (base peak, M+), 214 (M+2), and 216 (M+4) are characteristic of a molecule containing 3 chlorine atoms, although the peak expected at M/Z 218 (M+6) was not observable. Major peaks were observed at M/Z 178 (loss of H³⁵ Cl from 214), M/Z 176 (loss of H³⁵ Cl from 212, H³⁷ Cl from 214), M/Z 150 (loss of CO from 178), and M/Z 148 (loss of CO from 1976).

Similarly, PcpB converted both 2,4,6-trichlorophenol and 2,6-dichlorophenol to compounds with the same HPLC retention time as authentic 2,6-dichloro-p-hydroquinone. 2,6-dibromophenol, 2,4,6-tribromophenol, 2,6-dibromo-4-nitrophenol, and 3,5-dibromo-4-hydroxybenzonitrile were converted to end products sharing the same HPLC retention time. The end product from one of these compounds, 3,5-dibromo-4-hydroxybenzonitrile, was analyzed by GC/MS and confirmed to be 2,6-dibromo-p-hydroquinone (Topp et al., 1991).

PcpB quantitatively released an amino group as hydroxylamine (not as ammonium), a nitro group as nitrite, a cyano group as cyanide, and iodine as an iodide from the para position of substituted phenols tested. These results show that the enzyme not only catalyzes dehalogenation, but also removed hydrogen, nitro, amino, and cyano groups from benzene rings at the para position (in relation to the hydroxyl group of phenol). FIG. 9 shows proposed reaction equations for several representative substrates of PcpB.

Identification and Purification of PcpC

Identification of PcpC

As described above, the first step of PCP breakdown by Flavobacterium sp. Strain ATCC 39723 is an oxidation catalyzed by the enzyme PCP hydroxylase, also known as PcpB, which results in the formation of TECH. This reaction requires the presence of NADPH and oxygen. Whole cell studies with blocked mutants have been reported which propose that the Flavobacterium sp. reductively converts TeCH to 2,3,6-trichloro-p-hydroquinone (TrCH) and then to 2,6-dichloro-p-hydroquinone (DiCH), intermediates which are subsequently broken down by as yet uncharacterized reactions (Steiert and Crawford, 1986). The reductive dehalogenation of TeCH to TrCH and then to DiCH by a cell extract from ATCC 37923 has been confirmed by Xun et al. (1992b), herein incorporated by reference. A requirement for the reduced form of glutathione as a reducing agent for this reaction was also demonstrated.

The enzyme catalyzing the reductive dehalogenization of TeCH to DiCH is hereby termed tetrachlorohydroquinone reductase or PcpC. A method for purifying PcpC is described below.

Purification of PcpC

The enzymatic activity of PcpC was detected by the production of TrCH and DiCH from the substrate TeCH using high-pressure liquid chromatography (HPLC) analysis as described below. The purification scheme comprised seven major steps: cell extraction, streptomycin sulfate treatment, ammonium sulfate fractionation, phenyl agarose chromatography, DEAE agarose chromatography, mono Q chromatography, and gel filtration chromatography. The enzyme activity was followed using HPLC. Standard techniques were as described for PcpA purification unless otherwise stipulated.

Growth of cells.

Flavobacterium sp. Strain ATCC 39723 was cultured and PCP degradation was induced as described above.

Enzymatic activity assay.

The reaction mixture contained 50 mM KPi buffer (pH 7.4), 0.4% Tween 20 (Sigma), 100 μM of TeCH, and 1 mM ascorbic acid. All the reactions were incubated at 23° C. in an anaerobic chamber containing an atmosphere of 85% N₂, 5% CO₂, and 10% H₂ (Coy Laboratory Products Inc., Ann Arbor, Mich.). The reactions were stopped by addition of an equal volume of acetonitrile to the reaction mixture. TeCH stock solution (20 mM) was prepared in absolute ethanol. NADPH and NADH stock solutions (10 mM) were freshly prepared in 1 mM Tris buffer (pH 13) with 1 mM dithiothreitol. The stock solutions of FAD 10 (10 mM), FMN (10 mM), and the reduced form of GSH (1M) were freshly prepared in distilled water before use.

Analytical methods.

DiCH was obtained from Aldrich Chemical Co. (Milwaukee, Wis.). TrCH was made by enzymatic hydroxylation of 2,3,6-trichlorophenol by PcpB. TeCH, TrCH and DiCH were analyzed by HPLC on a Nova-Pak C-18 column (3.9 by 150 mm) (Waters, Milford, Mass.) with an 11 mM H₃ PO₄ -acetonitrile gradient as described above. Maximum absorption at 285, 300, 310 nm was recorded by using a Waters 490E programmable multiwave-length UV detector connected to a Maxima NEC computer workstation (Waters). The retention times of TeCH, TrCH and DiCH were 8.9, 8.1 and 6.7 min., respectively. TeCH and DiCH in the reaction mixtures were identified and quantified by comparison of their retention time and peak areas with that of authentic standards, respectively. TrCH was extracted with diethyl ether from the reaction mixture and analyzed by gas chromatography/mass spectrometry (GC/MS) as described by Topp and Akhtar (1990). Since DiCH could not be extracted from the reaction mixture with ether, DiCH peak was collected from the HPLC analysis using 0.1% trifluoroacetic acid instead of H₃ PO₄ in the elution gradient. The collected DiCH was dried in a Speed-vac (Savant Instruments Inc., Farmingdale, N.Y.) and resuspended in a few microliters of ethyl acetate before GC/MS analysis (Top and Akhtar, 1990). GSH and the oxidized form of glutathione (GS-SG) were detected by the enzymatic method of Tietze (1969).

Purification Steps

All operations were performed at 4° C., and all buffers contained 1 mM dithiothreitol. When ammonium sulfate was added, the buffer also contained 1 mM EDTA. Ammonium sulfate saturation levels referred to a temperature of 25° C.

(i) Extraction of Cells. Frozen (-20° C.) cell paste of 10 g was thawed in water and suspended in 50 ml of 20 mM Tris HCl buffer (pH 8.0) with 5 mM EDTA. The slurry was passed through an Aminco French pressure cell (model 4-3398) twice at 12,000 lb/in². The product was centrifuged at 17,000×g for 10 min., and the supernatant was saved. The precipitate was resuspended in 10 ml of the buffer, passed through a French pressure cell twice, and centrifuged as described above. The supernatant was combined with the previous supernatant, and the precipitate was discarded.

(ii) Streptomycin Sulfate Treatment. A half gram of streptomycin sulfate was added to the supernatant with constant stirring for 3 min. The mixture was centrifuged at 17,000×g for 10 min., and the precipitate was discarded.

(iii) Ammonium Sulfate Fractionation. The supernatant was diluted to 100 ml with the same Tris buffer, and solid ammonium sulfate was added to the supernatant to 30% saturation with constant stirring. The pH of the solution was not adjusted. After 10 min. of stirring, the mixture was centrifuged at 17,000×g for 10 min., and the precipitate was discarded. Additional solid ammonium sulfate was added to 65% saturation with constant stirring. After 10 min., the mixture was centrifuged at 17,000×g for 10 min. The precipitate was saved, and the supernatant was discarded.

(iv) Phenyl Agarose Chromatography. The precipitate was suspended with 1.5 volume of the Tris buffer. The suspension was centrifuged (17,000×g for 10 min.), and the precipitate was discarded. The supernatant was loaded onto a phenyl agarose (Sigma) column (30 by 1.5 cm), previously equilibrated with the Tris buffer containing 17% ammonium sulfate. The enzyme was eluted with 300 ml (linear gradient) of 17-1-0% ammonium sulfate. The enzyme activity was eluted out of the column from 14 to 12% ammonium sulfate. The fractions were pooled, and the proteins were precipitated by adding ammonium sulfate to 70% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.).

(v) DEAE Agarose Chromatography. The precipitate was suspended with an equal volume of 20 mM Tris buffer (pH 8.0) and dialyzed against 1 liter of the same buffer for 1 hr. The protein solution was loaded onto a DEAE agarose (Sigma) column (30 by 1.5 cm) previously equilibrated with the same buffer. The enzyme was eluted with 300 ml (linear gradient) of 0-to-300 mM NaCl in 20 mM Tris buffer (pH 8.0). The enzyme activity was eluted out of the column from 115 to 150 mM of NaCl. The fractions containing the enzyme activity were pooled, and the proteins were precipitated by adding ammonium sulfate to 70% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.).

(vi) Mono Q Chromatography. The proteins were resuspended in 1 ml of 20 mM Tris buffer (ph 8.0) and dialyzed against 1 liter of the same buffer for 2 hr. The protein solution was injected onto a Mono Q HR 5/5 column (Pharmacia) equilibrated with 20 mM Tris buffer. Proteins were eluted with a linear gradient of sodium chloride (percentage of 1M NaCl in a volume of 20 mM Tris: 0%, 2 ml; 0-20%, 20 ml; 100%, 4 ml; 0%, 2 ml) by a Pharmacia Fast Protein Liquid Chromatography system (Pharmacia LKB Programmer GP-250 Plus and P-500 pump). A single PcpC activity peak was associated with a peak at NaCl concentration of approximately 120 mM corresponding to fractions 14, 15 and 16 as shown in FIG. 10. The fractions containing enzyme activity were pooled and precipitated by adding ammonium sulfate to 70% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.).

(vii) Gel Filtration Chromatography. The protein precipitate was dissolved with an equal volume of Tris buffer and injected onto a Superose 12 column (Pharmacia) equilibrated with 20 mM Tris buffer containing 150 mM NaCl. The enzyme was eluted with the same buffer by the Pharmacia FPLC system. PcpC activity was primarily associated with a major protein peak with a retention volume of 13.5 ml, corresponding to fraction 14 shown in FIG. 11. The fractions containing the enzyme activity were pooled and precipitated by adding ammonium sulfate to 70% saturation. The precipitate was collected by centrifugation (17,000×g for 10 min.). The enzyme was resuspended in equal volume of 20 mM Tris buffer (ph 8.0) and stored at -20° C.

FIG. 12 shows SDS-PAGE of the products of the final two steps in PcpC purification: lane 1, fraction 15 from the gel filtration column of (vii) above; lane 2, molecular weight size markers (66, 45, 34, 24, 18.4, 14.3 kDa. from top to bottom); lane 3, fraction 14 from the gel filtration column of (vii) above; lane 4, empty; lane 5, molecular weight size markers as lane 2; lane 6, combined fractions 14, 15, and 16 from the Mono Q column of (vi) above. On the basis of this SDS-PAGE, the final purified PcpC preparation comprises a single band of approximately 26-29 kDa. (i.e., a molecular weight of 26,000-29,000).

Gene Cloning

The purification of proteins PcpA, PcpB and PcpC as described above facilitated the cloning of the genes encoding these three enzymes. The genes encoding the proteins PcpA, PcpB and PcpC are designated pcpA, pcpB and pcpC, respectively. The N-terminal amino acid sequence of each of the purified enzymes was determined on an Applied Biosystems Automated Protein Sequenator (Applied Biosystems, Foster City, Calif.). Based on these N-terminal amino acid sequences, degenerate oligonucleotide probes were synthesized on an Applied Biosystems DNA synthesizer and employed in the schemes described below in order to clone the genes encoding these three proteins.

Cloning of the Gene Encoding PcpA

The N-terminal sequence (residues 1 to 23) of the purified protein PcpA was determined to be Met-Glu-Thr-Asn-His-Ile-Thr-Ser-Leu-His-His-Ile-Thr-Ile-Cys-Thr-Gly-Thr-Ala-Gln-Gly-Asp-Ile. This amino acid sequence is part of SEQ. ID No. 1 in the accompanying sequence listing.

Two degenerate oligonucleotide primers, LX-1B and LX-2B, were made corresponding to amino acid residues 1 to 7 [5'-AUGGA(A/G)AC(T/C/A/G)AA(T/C)CA(T/C)AT(T/C/A)AC-3'] and 23 to 19 [5'-(A/G/T)AT(A/G)TC(T/G/A/C)CC(T/C)TG(A/G/T/C)GC-3'], respectively. Primers LX-1B and LX-2B are set forth in SEQ. ID Nos. 7 and 8, respectively, in the accompanying sequence listing. The primers were made in opposing orientations so as to amplify the internal region in a polymerase chain reaction (PCR). The PCR reactions were performed using conventional techniques (Mullis and Faloona, 1987; Innis et al., 1990) and the thermostable DNA polymerase from Thermus aquaticus DNA Taq polymerase (Saiki et al., 1988).

PCR was performed in a Coy temperature cycler (Coy Laboratory Products, Inc., Ann Arbor, Mich.) with a temperature profile of 94-55-72° C. cycling for 30 sec, 1 min., and 1 min., respectively, for 35 cycles. A 68 bp fragment was amplified by PCR, labeled with [α-³² P] dCTP by random priming (Sambrook et al., 1989) and used as a probe in genomic DNA and colony hybridizations. Southern hybridization (Southern, 1975) revealed that a single 21 kb BamHI fragment of Flavobacterium sp. Strain ATCC 39723 genomic DNA hybridized with the probe.

A genomic library of ATCC 39723 was subsequently constructed as described by Xun and Orser (1991a). Total DNA was extracted from ATCC 39723 by the method of Birboim and Doly (1979) and purified through two cycles of cesium chloride-ethidium bromide density gradient centrifugation (Sambrook et al., 1989). DNA extracted in this manner was partially digested with BamHI and size fractionated to select for 18 to 25 kb fragments (Sambrook et al., 1989)., The cosmid vector pLAFR3 was prepared as described by Staskawicz (1987) and BamHI pLAFR3 arms were ligated to ATCC 39723 BamHI-digested DNA fragments. Ligated molecules were packaged in vitro with freeze-thaw and sonication extracts of bacteriophage lambda, prepared after heat induction of lysogen strains BHB 2688 and BHB 2690 according to Maniatis et al. (1982). Packaged molecules were transduced into E. coli HB101 as described by Maniatis et al. (1982). Transductants were selected on Luria agar plates supplemented with streptomycin and tetracycline. Hybridization was performed using the 68 bp fragment labeled with [α-³² P] dCTP by random priming as described by Sambrook et al. (1989). Several positive colonies were detected by hybridization with the 68 bp probe. One positive colony was selected, and this cosmid, containing pcpA, the gene encoding the protein PcpA, was designated pLX001. The cloned DNA was analyzed by different restriction enzymes in a Southern hybridization with the amplified DNA probe. A 5.5 kb EcoRI fragment, which hybridized to the 68 bp probe, was subcloned into pBluescript KS⁺ phagemid (pBS-KS⁺) (Stratagene, San Diego, Calif.), to create pLX101⁺ and then cloned into pBluescript KS⁻ (pBS-KS⁻) (Stratagene, San Diego, Calif.) in the same orientation, to create pLX101⁻. An internal AccI fragment within the 5.5 kb EcoRI fragment was deleted by AccI digestion followed by religation. Subsequently, a 2.4 kb EcoRI-AccI fragment which hybridized to the 68 bp probe and contained pcpA was cloned into pBS-KS⁺ to produce pLX201⁺, and this fragment was also cloned into pBS-KS⁺ to produce pLX201⁻.

Nucleotide Sequence of pcpA

DNA sequencing was performed by the dideoxy-chain termination method of Sanger et al. (1977), using [α-³⁵ S] dATP and the M13 universal or reverse primer (Messing et al., 1981). Single-stranded DNA templates were recovered from pBluescript phagemid subclones by using helper phage as recommended by the vendor (Stratagene, La Jolla, Calif.). Nesting deletions were generated from both ends of the 2.4 kb EcoRI-AccI fragment by the procedures of Henikoff (1987), using mung bean nuclease and exonuclease III.

The management of compiled sequence data and DNA sequence analysis were done by using the IBI Pustell sequence analysis software (International Biotechnology Inc., New Haven, Conn.). Comparisons of nucleotide sequences and translated sequences were performed by using PC Gene software, version 6.1 (Intelligenetics, Mountain View, Calif.).

The 2.4 kb EcoRI-AccI fragment has been sequenced in both orientations. The pcpA sequence is presented as Sequence ID No. 1 in the Sequence Listing which forms part of this document. The open reading frame of the pcpA gene is 816 nucleotides in length from the GTG initiation codon (at base pairs 194-196 as shown in Sequence ID No. 1) to the TGA termination codon (at base pairs 1007-1010 as shown in Sequence ID No. 1).

The nucleotide sequence matched the N-terminal amino acid sequence perfectly. FIG. 13 shows the 20 N-terminal amino acids of PcpA. The sequence of these amino acids was determined by protein sequencing and confirmed by theoretical translation of the nucleotide sequence of pcpA. The predicted translation product of pcpA was 271 amino acids in length with a predicted molecular weight of 30,000, which is in good agreement with the protein purification data for PcpA. The computer-predicted translation product was hydrophilic with an isoelectric point of 5.75. A database search of the pcpA sequence has revealed no extensive sequence similarity with any characterized genes, although pcpA shared 53% homology with several mammalian cytochrome P450-type monooxygenases (Ishida et al., 1988) and the Pseudomonas putida catechol dioxygenase gene (Frantz and Chakrabarty, 1987). Protein database searches did not produce any significant results with these or other translated genes.

Cloning of the Gene Encoding PcpB

The N-terminal sequence (residues 1 to 13) of the purified protein PcpB were determined to be Ser-Thr-Tyr-Pro-Ile-Asn-Ala-Pro-Gly-Gln-Ser-Ala-Asp. This amino acid sequence is encompassed in SEQ. ID. No. 5 in the accompanying sequence listing. From the N-terminal sequence, a 37 mer degenerate primer was designed, corresponding to amino acid residues 2 to 13, for use as a hybridization probe for identification of the corresponding gene, pcpB. The primer was designated LX-6 and had the following 5' to 3' sequence: AC(C/G)TA(T/C)CC(C/G)AT(C/T)AA(T/C)GC(G/C)CC (C/G)GG(G/C)CA(G/A)(A/T)(G/C)(C/G)GC(G/C)GA(C/T)I, where I is inosine. The sequence of primer LX-6 is set forth in SEQ. ID. No. 9 of the accompanying sequence listing. DNA-DNA hybridization by Southern blot analysis (Southern, 1975) using [³² P]-randomly labeled primer LX-6 against EcoRI digested total genomic DNA from ATCC 39723, revealed a single 3.0 kb fragment. Hybridization was performed in 6xSSC solution (0.9M NaCl) (Sambrook et al., 1989) at 65° C., with subsequent washing of the filters under the same conditions. These conditions equate to a theoretical stringency of approximately 94% according to the formulae and calculations presented in Sambrook et al., (1989); that is, hybridization would only occur between molecules with sequence mismatches totaling no more than 6%. It should be noted that this is a theoretical calculation only (Sambrook et al. 1989). The LX-6 hybridizing fragment was cloned by electroelution of DNA from a gel fragment (Sambrook et al., 1989) which spanned the 3.0 kb region, followed by ligation to EcoRI digested pBluescript KS⁺ vector DNA, which encodes ampicillin resistance. Amp^(R) E. coli transformants containing the specific fragment were identified by colony hybridizations. One such positive clone was designated CO221, and its corresponding hybrid construct was designated pCO221.

Nucleotide Sequence of pcpB

The 3.0 kb EcoRI fragment was sequenced by the methods described above for pcpA, and a major open reading frame representing the pcpB gene was identified. The DNA sequence of pcpB is presented in Sequence ID No. 2 in the Sequence Listing which forms part of this document. The open reading frame of the pcpB gene is 1617 nucleotides inn length from the ATG initiation codon (at base pairs 328-330 as shown in Sequence ID No. 2) to the TGA termination codon (at base pairs 1942-1944 as shown in Sequence ID No. 2). The nucleotide sequence of this open reading frame matched the N-terminal amino acid sequence of the purified PcpB protein with the exception of the N-terminal methionine which was absent from the protein sequence determined by amino acid sequencing. This may indicate that this methionine is cleaved from the mature form of the protein. The predicted translation product of pcpB is 538 amino acids in length with a predicted molecular weight of approximately 59,900. The discrepancy between this molecular weight, the molecular weight of 63,000 as estimated by SDS-PAGE and the molecular weight of 66,000 determined by gel filtration chromatography reflects the diversity of experimental methods utilized to estimate the true molecular weight. PcpB is therefore a protein with a molecular weight in the range 59,000- 63,000. FIG. 13 shows the 20 N-terminal amino acids of PcpB. Amino acids 1-13 were determined by protein sequencing and confirmed by theoretical translation of the nucleotide sequence of pcpB. Amino acids 14-20 were determined by theoretical translation of the pcpB nucleotide sequence.

Cloning of the Gene Encoding PcpC

The N-terminal sequence (residues 1 to 18) of the purified protein PcpC were determined to be Pro-Glu-Val-Ser-Leu-Tyr-Asn-Tyr-Thr-Met-Ser-Ile-xxx-Ser-Met-Lys-Thr-Arg. Arg. -xxx- represents an amino acid which was unidentifiable by this protein sequencing. This amino acid sequence is set forth in SEQ. ID. No. 10 in the accompanying sequence listing. From the N-terminal sequence a 36mer degenerate primer was designed, corresponding to amino acid residues 1 to 12, as a hybridization probe for identification of the corresponding gene, pcpC. The primer, LX-7, was designed to be the complement to the mRNA so that it could be used for identifying the pcpC gene and mapping the transcriptional start site. The 5' to 3' sequence of LX-7 is as follows: GAT(C/G) (C/G) (T/A) CAT(G/C)GT(A/G)TAGTT (A/G)TA(C/G)AG(C/G) (C/G) (T/A) (G/C)AC(T/C)TC(G/C)GG. The sequence of primer LX-7 is set forth in SEQ. ID. No. 11 in the accompanying sequence listing.

LX-7 was labeled by random priming with [α-³² P] dCTP and used to probe EcoRI-digested genomic DNA from ATCC 39723. A 0.7 kb EcoRI fragment hybridized. This fragment was cloned as described above for pcpB in the vector pBluescript KS+, and the resulting recombinant replicon was designated pCO301. It was subsequently confirmed that this fragment is also contained in the cosmid clone, pLX001, which was originally identified as containing pcpA. Therefore, pcpA and pcpC reside in close proximity to each other on the Flavobacterium genome.

The 0.7 kb EcoRI fragment was partially sequenced and determined to contain only 100 bp of the coding sequence for pcpC (see below). Therefore, the 0.7 kb fragment was subsequently used in Southern hybridizations to identify a 9.0 kb EcoRV fragment from pLX001 which was subcloned to a 4.0 kb EcoRV-PstI fragment in the vector pBluescript KS+ and designated pC0303.

Nucleotide Sequence of pcpC

The sequence of the pcpC gene is presented as Secquence ID No. 3 in the Sequence Listing which forms part of this document. The open reading frame of the pcpC gene is 753 nucleotides in length from the ATG initiation codon (at base pairs 388-390 as shown in Sequence ID No. 3) to the TGA termination codon (at base pairs 1138-1140 as shown in Sequence ID No. 3). The predicted translation product of pcpC is 250 amino acids in length with a predicted molecular weight of approximately 28,500. The 5' nucleotide sequence obtained matches the N-terminal amino acid sequence with the exception of the N-terminal methionine which was not detected by protein sequencing. A similar observation was reported for PcpB, as discussed above. The single amino acid which was unidentifiable by protein sequencing is now shown to be a cysteine by a theoretical translation of the obtained DNA sequence. FIG. 13 shows the 20 N-terminal amino acids of PcpC. Amino acids 1-12 and 14-18 were determined by protein sequencing and confirmed by theoretical translation of the pcpC nucleotide sequence. Amino acids 13, 19 and 20 were determined by theoretical translation of the pcpC nucleotide sequence.

Discussion

Having herein presented purified preparations of PcpA, PcpB and PcpC and cloned DNA molecules which encode these proteins, proteins and DNA molecules which possess the biological characteristics of the specific proteins and DNA molecules presented are also comprehended by this application.

It will be appreciated by one skilled in the art that this invention, by providing cloned DNA molecules and nucleotide sequences, facilitates the synthesis of many variations on the disclosed DNA molecules and nucleotide sequences, through the use of standard molecular biology laboratory techniques. Such variant DNA molecules include those created by standard DNA mutagenesis techniques, for example, M13 primer mutagenesis, details of these techniques are provided in Sambrook et al. (1989). By the use of such techniques, variants may be created which differ in minor ways from those disclosed. DNA molecules and nucleotide sequences which are derivatives of those specifically disclosed herein and which differ from those disclosed by the deletion, addition or substitution of nucleotides while still maintaining the essential characteristics of the encoded protein are comprehended by this invention. Also within the scope of this invention are small DNA molecules which are fragments of the disclosed DNA molecules. Such fragments of the disclosed DNA molecules include oligonucleotides suitable for use as hybridizations probes or polymerase chain reaction (PCR) primers. These fragments are preferably greater than 15 nucleotides in length when selected for use as hybridization probes or PCR primers. Most preferably they are greater than 20 nucleotides in length and most preferably they are greater than 25 nucleotides in length.

DNA molecules and nucleotide sequences which are derived from the disclosed DNA molecules as described above may also be defined as DNA sequences which hybridize under stringent conditions to the DNA sequences disclosed, or fragments thereof.

Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridizing DNA used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed by Sambrook et al. (1989), chapters 9 and 11, herein incorporated by reference. In preferred embodiments of the present invention, stringent conditions may be defined as those under which DNA molecules with more than 25% sequence variation (also termed "mismatch") will not hybridize. In a more preferred embodiment, stringent conditions are those under which DNA molecules with more than 15% mismatch will not hybridize, and more preferably still, stringent conditions are those under which DNA sequences with more than 10% mismatch will not hybridize. In a most preferred embodiment, stringent conditions are those under which DNA sequences with more than 6% mismatch will not hybridize. Hybridizations under the stringent conditions of the most preferred embodiment could be carried out under the hybridization conditions described herein with respect to the initial isolation of the pcpA, pcpB and pcpC genes and in Example 5.

DNA sequences which, but for the degeneracy of the genetic code, would hybridize to disclosed DNA molecules, and oligonucleotide sequences are also comprehended by this invention. The degeneracy of the genetic code further widens the scope of the present invention as it enables major variations in the nucleotide sequence of a DNA molecule while maintaining the amino acid sequence of the encoded protein. For example, the second amino acid residue of the PcpB protein is serine. This is encoded in the pcpB gene by the nucleotide codon triplet TCG. Because of the degeneracy of the genetic code, five other nucleotide codon triplets--TCT, TCC, TCA, AGT and AGC--also code for serine. Thus, the nucleotide sequence of the pcpB DNA could be changed at this position to any of these five codons without affecting the amino acid composition of the encoded protein or the characteristics of the protein. The genetic code and variations in nucleotide codons for particular amino acids is presented in Tables 3 and 4. Based upon the degeneracy of the genetic code, variant DNA molecules may be derived from the DNA molecules disclosed herein using standard DNA mutagenesis techniques as described above, or by synthesis of DNA sequences. DNA sequences which do not hybridize under stringent conditions to the DNA sequences disclosed by virtue of sequence variation based on the degeneracy of the genetic code are herein also comprehended by this invention.

                  TABLE 3                                                          ______________________________________                                         The Genetic Code                                                               First                          Third                                           Position                                                                              Second Position         Position                                        (5" end)                                                                              T         C       A       G     (3' end)                                ______________________________________                                         T      Phe       Ser     Tyr     Cys   T                                              Phe       Ser     Tyr     Cys   C                                              Leu       Ser     Stop (och)                                                                             Stop  A                                              Leu       Ser     Stop (amb)                                                                             Trp   G                                       C      Leu       Pro     His     Arg   T                                              Leu       Pro     His     Arg   C                                              Leu       Pro     Gln     Arg   A                                              Leu       Pro     Gln     Arg   G                                       A      Ile       Thr     Asn     Ser   T                                              Ile       Thr     Asn     Ser   C                                              Ile       Thr     Lys     Arg   A                                              Met       Thr     Lys     Arg   G                                       G      Val       Ala     Asp     Gly   T                                              Val       Ala     Asp     Gly   C                                              Val       Ala     Glu     Gly   A                                              Val (Met) Ala     Glu     Gly   G                                       ______________________________________                                          "Stop (och)" stands for the ocre termination triplet, and "Stop (amb)" fo      the amber. ATG is the most common initator condon; GTG usually codes for       valine, but it can also code for methionine to initiate an mRNA chain.   

                  TABLE 4                                                          ______________________________________                                         The Degeneracy of the Genetic Code                                             Number of                   Total                                              Synonymous                  Number of                                          Codons      Amino Acid      Codons                                             ______________________________________                                         6           Leu, Ser, Arg   18                                                 4           Gly, Pro, Ala, Val, Thr                                                                        20                                                 3           Ile              3                                                 2           Phe, Tyr, Cys, His, Gln,                                                                       18                                                             Glu, Asn, Asp, Lys                                                 1           Met, Trp         2                                                 Total number of codons for amino acids                                                                 61                                                     Number of codons for termination                                                                        3                                                     Total number of codons in genetic code                                                                 64                                                     ______________________________________                                    

One skilled in the art will recognize that the mutagenisis protocols described above will also facilitate the production of proteins which differ in certain structural aspects from either PcpA, PcpB or PcpC, yet which proteins are clearly derivatives of these named proteins and which maintain the essential characteristics of the parent protein. Newly derived proteins may also be selected in order to obtain variations on the characteristics of the parent protein, as will be more fully described below. Such derivatives include those with variations in amino acid sequence including minor deletions, additions and substitutions.

While the site for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed protein variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence as described above are well known, for example, M13 primer mutagenesis.

Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. Obviously, the mutations that will be made in the DNA encoding the protein must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.

Substitutional variants are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table 5 when it is desired to finely modulate the characteristics of the protein.

                  TABLE 5                                                          ______________________________________                                         Original Residue  Exemplary Substitutions                                      ______________________________________                                         Ala               ser                                                          Arg               lys                                                          Asn               gln, his                                                     Asp               glu                                                          Cys               ser                                                          Gln               asn                                                          Glu               asp                                                          Gly               pro                                                          His               asn; gln                                                     Ile               leu, val                                                     Leu               ile; val                                                     Lys               arg; gln; glu                                                Met               leu; ile                                                     Phe               met; leu; tyr                                                Ser               thr                                                          Thr               ser                                                          Trp               tyr                                                          Tyr               trp; phe                                                     Val               ile; leu                                                     ______________________________________                                    

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 5 i.e. selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

The effects of these amino acid substitutions or deletions or additions may be assessed for derivatives of PcpB and PcpC by analyzing the enzymatic characteristics of the derivative proteins using the enzyme assays for PcpB and PcpC disclosed herein.

The purified enzymes and the DNA molecules encoding the enzymes presented herein may be utilized in aspects of both the study and practical application of microbial catabolism of PCPs and other halogenated and otherwise substituted aromatics.

Utilities of the present invention include, but are not limited to, those utilities described in the following examples.

Those skilled in the art will recognize that the utilities herein described are not limited to the specific experimental modes and materials presented and will appreciate the wider potential utility of this invention.

EXAMPLE 1

Purified proteins PcpB and PcpC may be used for the in vitro dechlorination of PCP. Both of these proteins remove chlorine atoms from their substrate molecule, thus PcpB dechlorinates PCP to yield TeCH and PcpC dechlorinates TeCH to yield TrCH and DiCH. These proteins may be purified from the Flavobacterium as described above or from microorganisms transformed with the recombinant DNA vectors and which, by virtue of being so transformed, produce PcpB and/or PcpC. Suitable transformed microorganisms include the disclosed E. coli clones which are transformed with recombinant DNA vectors pCO221 and pCO303. These vectors encode PcpB and PcpC, respectively. Alternatively, as described in Example 2, the pcpA, pcpB or pcpC genes may be cloned into vectors which are designed to promote the high-level expression cloned genes. Proteins produced in these microorganism are purified according to the methodologies described for the purification of the proteins from the Flavobacterium. Minor variations in these methodologies to take account of the characteristics of a particular chosen microorganism may be required.

Conditions suitable for the conversion of PCP to TeCH by PcpB and for the conversion of TeCH to TrCH and DiCH by PcpC are given above and may be adapted according to the scale of the desired reaction. An embodiment of the current invention is a composition comprising both purified PcpB and PcpC which is used for the conversion of PCP to DiCH.

Purified PcpB may also be utilized to degrade a range of other compounds as disclosed above.

The use of these purified proteins for the degradation of PCP may involve other embodiments including immobilized enzyme formats in which the purified enzymes are attached to an insoluble surface. Methods suitable for the attachment of proteins such as PcpB and PcpC to insoluble surfaces are well known in the art.

EXAMPLE 2

As described in Example 1, purified PcpB and PcpC may be used for the in vitro dechlorination of PCP. The pcpB and pcpC gene may be cloned into high-level expression vectors in order to promote the levels of PcpB and PcpC protein production. The following example illustrates the use of a high-level expression vector to obtain expression of PcpB in E. coli. In order to obtain high-level expression of the pcpB gene, the gene was excised from the pCO221 vector described above by digestion with the restriction enzymes HpaI and HindIII. HpaI cleaves in the middle of the hexanucleotide GTTAAC, leaving a blunt-ended fragment. This recognition site is present in the pcpB gene sequence at base pairs 211-216 upstream of the ATG initiation codon, as shown in Sequence ID No. 2. The HindIII recognition site is present in the polylinker region of pCO221 located downstream of the pcpB gene. This HpaI-HindIII fragment, therefore, contains the entire pcpB gene. This fragment was cloned into the vector pKK223-3, an expression vector available from Pharmacia LKB Biotechnology, Inc. (Piscataway, N.J.). The pKK223-3 vector was digested with the restriction enzymes SmaI and HindIII. Digestion with the restriction enzyme SmaI leaves a blunt-ended fragment which is compatible with the blunt-ended fragment produced by digestion with HpaI. The HpaI-HindIII pcpB gene fragment was therefore ligated into the SmaI-HindIII digested pKK223-3 vector to create the plasmid pCL3. The construction is shown schematically in FIG. 14. This construction results in the ATG initiation codon of the pcpB gene being located adjacent to the P_(tac) promoter of the pKK223-3 vector. The orientation of the p_(tac) promoter is such that it will drive high-level expression of the pcpB gene when the vector is transformed into E. coli. For the above manipulations, restriction endonucleases, T4 DNA ligase, random-primed DNA labeling kit and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from United States Biochemical Corp., Cleveland, Ohio, and used as recommended by the supplier.

Following electroporation of E. coli, JM 105 with 1 ml of the ligation mixture, and colony hybridization with ³² P-dCTP-labeled probe derived from the pcpB gene, the clone CL3 was picked from a large number of negative colonies in the background. The structure of this clone was confirmed to be the structure shown in FIG. 14 by plasmid miniprep from 5 μl overnight culture and digestion with EcoRI.

The production of PcpB in the transformed E. coli containing pCL3 was assessed by SDS-PAGE and immunoblotting. Cells were grown in Luria broth with and without the presence of IPTG. Samples were taken from the cultures at hourly intervals and crude proteins extracted from these samples were examined by SDS-PAGE (10% acrylamide) using a Bio-Rad Mini-Protean II apparatus and quantified by the Lowry assay (Lowry et al., 1951). The SDS-PAGE gels were stained with Coomassie Blue and the results of this are shown in FIG. 15, Panel A. The protein molecular weight standards shown in FIG. 15, Panel A, were purchased from Bethesda Research Laboratories, Inc. (Gathersberg, Md.) and are, in descending order: 205, 116, 97, 66, 45 and 29 kDa. Purified PCP hydroxylase (PcpB protein) was loaded into the lane adjacent the molecular weight standards. Immunoblotting of the SDS-PAGE was performed by a slight modification of that described by Bollag and Edelstein (1991) using a Bio-Rad Mini-Transblot Electrophoretic Transfer Cell. Nonspecific binding of antibody was blocked by a one-hour incubation in 3% gelatin at 37° C. with gentle shaking. A 1:1000 dilution of rabbit polyclonal antibody raised against the purified PcpB protein (prepared commercially by Berkeley Antibody Co., Berkeley, Calif.) was used as the primary antibody to detect the PcpB protein. The bound anti-PcpB antibody was detected with goat anti-rabbit antibody conjugated with alkaline phosphatase, supplied by Tago, Inc. (Burlingame, Calif.) and blot development was performed as described by the manufacturers. The immunoblot resulting from this procedure is shown in FIG. 15, Panel B. The blot confirms the expression of PcpB by the transformed E. coli cells.

The activity of the PcpB protein expressed in the transformed E. coli was then determined by enzyme assay. E. coli JM 105 containing pCL3 was cultured in Luria broth (Maniatis et al., 1992) containing 50 mg/liter ampicillin. The growth of the culture was followed spectrophotometrically, and when the OD₆₀₀ of the culture reached 0.7, IPTG was added to a final concentration of 1 mM. Two to three hours after the addition of IPTG, the culture was centrifuged, and the cells were collected. Cells from one liter of the growth medium were used to prepare cell extracts as described previously for Flavobacterium (Xun and Orser, 1991c.). Forty to 60% ammonium sulphate fractions were dialyzed for four hours against 0.75 liters of 20 mM Tris-5 mM EDTA solution, pH 8.0, with stirring at 4° C., reprecipitated with 60% ammonium sulphate and then subjected to enzyme assays.

Enzyme assays were performed as described above for the PcpB protein extracted from Flavobacterium, using a Waters HPLC with a Nova-pak C18 column with an H₃ PO₄ -acetonitrile gradient. FIG. 16 shows the results of this HPLC analysis: proteins extracted from E. coli transformed with pCL3 and induced with IPTG converted substrate PCP to TeCH, whereas control cells (E. coli transformed with pKK223-3 and induced with IPTG) showed no such activity. This experiment confirmed the activity of the pcpB protein in the transgenic E. coli cells.

These experiments confirmed that functional PcpB protein was indeed expressed by the recombinant E. coli strain transformed with pCL3.

EXAMPLE 3

The cloned DNA molecules which include genes pcpA, pcpB and pcpC which encode the enzymes PcpA, PcpB and PcpC, respectively, may be utilized in the creation of transgenic microorganisms which possess enhanced PCP dechlorination or degrading ability. Such microorganisms could be used in bioreactors wherein PCP containing material, supplemented with appropriate nutritional additives, would be degraded by these organisms. Alternatively, these microorganisms could be applied directly onto contaminated environmental sites, including PCP contaminated soils and bodies of water. Yet another use for such microorganisms would be the production of the purified proteins. Thus, if these microorganisms were more readily cultured than the Flavobacterium or produced higher levels of the proteins, then such organisms would be preferred for the large scale purification of the proteins. Transformed microorganisms developed according to this invention may be transformed with vectors including one or more of the genes pcpA, pcpB and pcpC. Thus, transformed microorganisms may be transformed with one of the genes selected from the group consisting of pcpA, pcpB and pcpC, or combinations of two of the genes selected from this group or, alternatively, all three of the genes of this group.

The disclosed DNA molecules encoding PcpA, PcpB or PcpC may be transformed into E. coli on recombinant vectors, such as vectors based on the cosmid vector pLAFR3 or the phagemid pBluescript as disclosed, or into other microorganisms using the disclosed and/or other vectors and in association with other DNA sequences such as promoters which effect high level expression of the genes. Microorganisms potentially suitable as hosts include, but are not limited to, soil bacteria such as Pseudomonas spp. for in situ application and Bacillus spp. and E. coli for bioreactor and protein purification purposes.

In general, plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts. The vector ordinarily carries a replication site as well as marker sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar et al. (1977)), pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA construction.

Promoters suitable for use with prokaryotic hosts illustratively include the β-lactamase and lactose promoter systems (Chang et al. (1978); Goeddel et al. (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (Goeddel (1980) and EPO Appln. Publ. No. 36,776) and hybrid promoters such as the tac promoter (H. de Boer et al. (1983)). However, other functional bacterial promoters are suitable. Their nucleotide sequences are generally known, thereby enabling a skilled worker operably to ligate them to DNA encoding PcpA, PcpB or PcpC (Siebenlist et al. (1980)) using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding growth hormone receptor and binding protein.

Those skilled in the art will recognize that the creation of such transgenic microorganisms is now enabled by this invention and that this approach is not limited to the specific experimental modes and materials described and will appreciate the wider potential utility of this invention.

EXAMPLE 4

Transgenic microorganisms producing the PcpB or PcpC proteins may also be used for the dechlorination of PCP without further purification of the PcpB or PcpC proteins. Thus, the dechlorination of PCP to TeCH may be effected by exposing PCP directly to microorganisms transformed with DNA that encodes the PcpB protein. Furthermore, the TeCH produced as a result of the dechlorination of PCP may be further dechlorinated to TrCH and DiCH by exposure to microorganisms transformed with DNA that encodes the PcpC protein. Alternatively, the dechlorination of PCP to DiCH may be achieved by exposing PCP to microorganisms that are transformed with DNA that encodes both the PcpB and PcpC proteins. The following example serves to illustrate that PcpB activity is exhibited by whole cells of E. coli transformed with the pcpB gene.

E. coli JM105 transformed with pCL3 and E. coli JM105 transformed with pKK223-3 (as a control) produced as described above were cultured in Luria broth supplemented with ampicillin (50 μg/ml) and IPTG (1 mM) at room temperature with shaking. The growth of the culture was followed spectrophotometrically and when the OD₆₀₀ of the culture reached 0.7 (mid-log phase), the cells were pelleted by centrifugation. The cell pellet was then resuspended in minimal medium (M9) supplemented with thiamine, ampicillin (50 μg/ml), IPTG (1 mM), PCP (0.3 mM or 80 PPM) and 10 mM phosphate buffer (pH 7.0) with subsequent gentle shaking at room temperature. As shown in Table 6 below, at specified time points, 600 μl of the culture was removed, the cells pelleted out, and the supernatant evaluated spectrophotometrically at 318 nm.

                  TABLE 6                                                          ______________________________________                                         Time       JM105 (pCL3)                                                                               JM105 (pKK223-3)                                        ______________________________________                                         10 min     1.3675      0.8718                                                  20 min     0.8179      0.8210                                                  30 min     0.9678      0.6407                                                  40 min     0.6287      0.5354                                                  50 min     0.6562      0.5915                                                  60 min     0.6414      0.5707                                                  70 min     0.6223      0.7768                                                  80 min     0.6448      0.5487                                                  120 min    0.6448      0.5486                                                  660 min     0.15913    0.0471                                                  ______________________________________                                    

PCP absorbs at 318 nm and therefore the measurements shown in Table 6 above were intended to determine whether the E. coli cultures were removing PCP from the growth medium. Both the JM105 transformed with pCL3 and JM105 transformed with the vector pKK223-3 produced spectrophotometric results which indicated the removal of PCP from the culture. The decrease in absorbance at 318 nm for both cultures is likely explained by the uptake of PCP into the E. coli cells. It is proposed that this occurs because of the lipophilic nature of the PCP and that this uptake is independent of the presence of the particular DNA molecules transformed into the E. coli cells.

HPLC analysis was employed to confirm the dechlorination of PCP by the intact cell culture of E. coli transformed with pCL3. Samples were removed from the cultures at the 660 minute time point and 20 μl of cells were mixed with 20 μl of acetonitrile. The resulting lysed cellular debris was pelleted out by centrifugation and the supernatant was loaded onto a C18 reverse phase HPLC column, as described above. The resulting chromatograms are shown in FIG. 17. This experiment confirms that PCP breakdown to TeCH did occur in E. coli cells transformed with pCL3 but that no such dechlorination occurred in the control E. coli culture.

These experiments confirm the ability of intact E. coli cells transformed with the pcpB gene to dechlorinate PCP to TECH.

EXAMPLE 5

The purified proteins PcpA, PcpB and PcpC may also be employed to prepare monoclonal antibodies (MAbs). Methods for making MAbs are well known in the art and are described in publications such as that by Harlow and Lane (1988).

The production of PcpA and PcpB by Flavobacterium sp. Strain ATCC 39723 are induced by the presence of PCP. This finding can be exploited to develop a novel immunoassay method by which the levels of PCP present in a sample taken from a contaminated environmental site may be determined.

MAbs raised against PcpA (or PcpB) are used to determine the levels of PcpA (or PcpB) produced by the organism in response to the presence of PCP in a given sample thereby allowing quantification of PCP in the sample. The following example describes the use of such MAbs raised against PcpA. PcpA is selected as the preferred embodiment because it is produced in the periplasm and is therefore most easily and completely extracted from the cells.

PcpA is purified by the methodology described above. MAbs against the purified protein may be raised in a number of animals, including mice. Methods of raising MAbs include, but are not limited to, the approach described herein: a number of mice (typically 2-6) are injected with a dose of the purified protein (typically 50 μg) as part of an antigen preparation (which may include other compounds such as Freund's adjuvant). Approximately two weeks after this primary immunization, the mice receive a second, booster, inoculation, and thereafter test bleeds are performed on a regular basis to assess the development of the antibody response to PcpA within the mice. Suitable methods for determining the anti-PcpA antibody titer include, but are not limited to, the antibody capture dot blot assay, as described in Harlow and Lane (1988).

In one embodiment of this assay, nitrocellulose paper is incubated in a solution of purified PcpA (at a concentration of 10-50 μg/ml) so as to allow the protein to bind to the paper. Bound protein is subsequently fixed to the paper by incubation in 0.02% sodium azide. Serum extracted from the immunized mice is serially diluted and 1 μl samples of each dilution are spotted onto the paper. Unbound antibodies are removed by washing and the presence of mouse anti-PcpA antibodies attached to the bound PcpA is then detected using ¹²⁵ I-labeled rabbit anti-mouse immunoglobulin. The antibody titer is determined by the highest dilution of the serum which shows the presence of anti-PcpA antibodies.

Further booster inoculations are provided as necessary in order to raise the anti-PcpA titer. A mouse with a high anti-PcpA titer is selected for hybridoma production. Antibody-secreting cells are prepared from the mouse, mixed with myeloma cells and fused. Following fusion, the resultant hybridoma cells are diluted in selective medium and plated in multi-well culture dishes. The hybridomas are screened for the production of anti-PcpA antibodies and cells from positive wells are single-cell cloned. MAbs produced from these cloned cell lines may be harvested from tissue culture supernatants or ascitic fluid.

In order to assess the level of PCP contamination at a given environmental site, samples taken from the site are serially diluted and the diluted samples incubated with cells of Flavobacterium sp. Strain ATCC 39723. Following an incubation period of preferably not less than 20 minutes to allow induction of PcpA, the samples are treated to release induced PcpA. Suitable treatments for the release of PcpA from the periplasmic space of the bacteria are described earlier in this work.

The levels of induced PcpA are then determined by immunoassay using MAbs raised against PcpA. Various methods for such an immunoassay are well known in the art (Harlow and Lane, 1988). A version of the antibody capture assay may be employed for this purpose. In one embodiment of this assay, samples of the protein released from the periplasmic space of the treated cells are spotted onto nitrocellulose paper. The paper is then incubated firstly with anti-PcpA MAbs and then with ¹²⁵ I labeled rabbit anti-mouse immunoglobulin. Exposure of the treated nitrocellulose to X-ray film subsequently allows the presence of PCP in the original sample to be detected. The amount of PCP in each sample is determined in reference to test standards created by adding known amounts of PCP to cells and then following the above procedure. Levels of PCP in particular samples are then calculated from those highest dilutions of each sample which give a positive induction response.

EXAMPLE 6

Monoclonal antibodies to each of the purified proteins can be used to assess the levels of PcpA, PcpB or PcpC production by new isolates of PCP degrading Flavobacteria or other species under a variety of conditions. MAbs may be used to quantitate the production of these proteins by a variety of methods, including antibody capture assays as described in Example 2 and by Harlow and Lane (1988) (chapter 14), which is incorporated by reference. This type of assay can be used to determine optimal conditions for PCP breakdown by providing quantitation of the levels of these proteins under a variety of conditions. Conditions which result in elevated levels of PcpA, PcpB and PcpC are optimal for PCP breakdown activity.

EXAMPLE 7

The cloned genes, or portions of these genes, may be used as probes to clone homologous genes from other microorganisms which display the ability to degrade PCP, halogenated aromatics or other substituted aromatics. In this way, genes which encode enzymes exhibiting differing substrate specificities may be obtained which, in turn, will widen the range of environmental contaminants that can be treated by novel bioremediation approaches.

By way of demonstration of the utility of the genes for this purpose, DNA was extracted from four microorganisms with known PCP breakdown activity (Flavobacterium ATCC 39723, Pseudomonas SR3, Arthrobacter ATCC 33790 and Rhodococcus chlorophenolicus) and a related fifth organism (Arthrobacter DSM 20407) which lacks this activity. Pseudomonas SR3 was obtained from Dr. Peter Chapman, United States Environmental Protection Agency, Florida Gulf Breeze Station, Rhodococcus chlorophenolicus was obtained from Dr. R. Crawford, University of Idaho and the other isolates were obtained from culture collections.

The DNAs were digested with EcoRI, electrophoresed and Southern blotted according to Sambrook et al. (1989). Southern blots were subsequently probed with [α-³² P] dCTP labeled probes derived from the cloned genes. Hybridization was performed in 6×SSC (0.9M NaCl) at 65° C., filters were washed in 0.3×SSC (0.045M NaCl) at 65° C. Hybridization results are tabulated in Table 4. Three of the four strains with known PCP degrading activity each showed a single hybridizing band with each of the three gene probes indicating the presence of homologous genes. DNA from Rhodococcus chlorophenolicus did not show hybridization under the stringent conditions used; it is proposed that the genes encoding enzymes of the PCP degrading pathway of this organism are less homologous to the gene probes than those of the other organisms. Hybridization under less stringent conditions may be used to detect genes with weaker homology. No hybridization was observed to the DNA of the control strain, Arthrobacter DSM 20407.

                  TABLE 7                                                          ______________________________________                                                     pcpA       pcpB        pcpC                                                    2.3 kb     2.3 kb      0.7 kb                                      Probe fragments                                                                            EcoRI/AccI EcoRI/BamHI EcoRI                                       ______________________________________                                         Microoorganism                                                                 Flavobacterium sp.                                                                         5.5 kb     3.0 kb      0.7 kb                                      ATCC 39723                                                                     Pseudomonas SR3                                                                            5.0 kb     3.0 kb      1.3 kb                                      Arthrobacter                                                                               5.0 kb     3.0 kb      1.3 kb                                      ATCC 33790                                                                     Arthrobacter DSM                                                                           --         --          --                                          20407                                                                          Rhodococcus --         --          --                                          chlorophenolicus                                                               ______________________________________                                    

The hybridizing DNA of these strains may be cloned and the PCP breakdown genes identified using the strategies outlined in this invention.

EXAMPLE 8

It is frequently desirable to assess contaminated environments for the presence of microbial populations capable of breaking down the particular contaminants present in these environments, in order to determine suitable decontamination strategies. In accordance with the present invention, a novel, highly sensitive method of detecting the presence of microorganisms capable of breaking down PCP is presented. Microorganisms which possess PCP breakdown activity and which thus carry DNA sequences homologous to either pcpA, pcpB or pcpC, may be detected by this method.

Southern hybridization, as described in Example 7, may be utilized directly to detect these organisms. However, these microbial populations are often not readily cultured in the laboratory and so such this approach may be unsatisfactory.

A combination of the polymerase chain reaction (PCR) and DNA hybridization offers a much more sensitive method for detection of such microorganisms; it does not require the culturing of the microorganisms and is theoretically capable of detecting a single microorganism in a given sample. The detection of other types of microorganisms in environmental samples using primers derived from other cloned genes is discussed by Atlas and Bej (1990) .

DNA primers suitable for use in the PCR may be derived from the sequences of either pcpA, pcpB or pcpC. Methods for designing primers are well known in the art and are disclosed in publications including Innis et al. (1990). The cloning of genes from other organisms as described in Example 7 above would allow the determination of highly conserved regions of these genes. The selection of primers from such highly conserved regions for use in the PCR would ensure the detection of a wide range of PCP degrading organisms by avoiding less well conserved sequence regions.

Environmental samples are treated to extract microorganisms and the organisms are lysed to release DNA. The samples are then subjected to the PCR in the presence of the appropriate primers. DNA sequences amplified by the PCR are then detected by Southern blotting or dot blotting using labeled DNA probes corresponding to the amplified DNA regions of the gene from which the probes were derived, as described by Atlas and Bej (1990). The detection of hybridizing, amplified DNA indicates the presence of PCP degrading microorganisms in the sample.

Having illustrated and described the principles of purifying the proteins, cloning the genes encoding these enzymes and modes of use of these purified proteins and their corresponding genes, it should be apparent to one skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications coming within the spirit and scope of the claims presented herein.

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    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 11                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1109 base pairs                                                    (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Double- stranded                                             (D) TOPOLOGY: Linear                                                           (ii) MOLECULE TYPE: Genomic DNA                                                (A) DESCRIPTION:                                                               (i ii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Flavobacterium sp. Strain ATCC 39723                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       CGAAGAAGCGCCGGAAAAGCCCAGTATAGCGCAACGCCTTCCAGCGCCAAGGCTATTC58                   ATTATTCGAATGCGGGATATTCGCGAACCTGCGTGGAGCA AGCACCCGTCCGATGCTATT118               TCAACAATGGTCCCGCCCCATTGCCGCAAGGTGGAAGAAGGGCGCGATTCTTCGGACTTA178                TCTTGGAGAGCGATCGTGGAAACGAACCATATCACCAGTCTGCATCAT226                            MetGluThrAsnHisIleTh rSerLeuHisHis                                             1510                                                                           ATCACCATCTGCACCGGCACGGCGCAGGGGGACATCGATTTCTTC271                               IleThrIleCysThrGlyThrAlaGlnGlyAspIleAspPhePhe                                  15 2025                                                                        GTGAAGGTCATGGGCCAGCGCTTCGTGAAGCGCACGCTGTTCTAT316                               ValLysValMetGlyGlnArgPheValLysArgThrLeuPheTyr                                  3035 40                                                                        GACGGCAGCATTCCCATCTATCACCTGTACTTCGCGGACGAACTG361                               AspGlySerIleProIleTyrHisLeuTyrPheAlaAspGluLeu                                  455055                                                                         GGCAC GCCCGGCACCGTCATGACCACCTTCCCCACCCGCCGCACC406                              GlyThrProGlyThrValMetThrThrPheProThrArgArgThr                                  606570                                                                         GGCCAGAAGGGGCGCAAGGGG TCCAACCAGTTCACCGTCTGCACC451                              GlyGlnLysGlyArgLysGlySerAsnGlnPheThrValCysThr                                  758085                                                                         TACGCCATCCCCAAGGGGTCGCTGGAATGGTGGATCGG CCATCTG496                              TyrAlaIleProLysGlySerLeuGluTrpTrpIleGlyHisLeu                                  9095100                                                                        AACGCGCACGGCATCGCGACCGGCGAGCCGGCACCCGTTTCGGCC541                                AsnAlaHisGlyIleAlaThrGlyGluProAlaProValSerAla                                 105110115                                                                      AGCGCTATGTGGGTTTCCAGCATCCGACTGCGGATCGATTTCGAG586                               SerAlaMETTrpValS erSerIleArgLeuArgIleAspPheGlu                                 120125130                                                                      GTGCTGGAGGATGAGAATGACACGCGTCAGCCCTATGACTCGCCC631                               ValLeuGluAspGluAsnAspThrArgGlnPr oTyrAspSerPro                                 135140145                                                                      TATGTCCCCATCGAGCATGCGCACGGCGGCTTCCATAGCTGGACG676                               TyrValProIleGluHisAlaHisGlyGlyPheHisSerTrpThr                                  15 0155160                                                                     GCGTCGGTCCGCGAGCTGGAGGACATGGACTTCTTCATGGAGAAT721                               AlaSerValArgGluLeuGluAspMetAspPhePheMETGluAsn                                  165170 175                                                                     TGCTGGAATTTCGAGAAGATCGGCGAGGAAGGCAACCGTCACCGC766                               CysTrpAsnPheGluLysIleGlyGluGluGlyAsnArgHisArg                                  180185190                                                                      TATCGCGTGAAGGGCACGACGGAATCGGGCACGATCATCGACCTG811                               TyrArgValLysGlyThrThrGluSerGlyThrIleIleAspLeu                                  195200205                                                                      CTGCATGAACCGGAC CGTCGTCAGGGTAGCTGGACCATCGCGGAA856                              LeuHisGluProAspArgArgGlnGlySerTrpThrIleAlaGlu                                  210215220                                                                      GGCATCATCCACCATGGCGCCTTCGCGGTG CCGGACATGGACATC901                              GlyIleIleHisHisGlyAlaPheAlaValProAspMetAspIle                                  225230235                                                                      CAGGCGCGCATCAAGTTCGAAACCGAGGGCGTCGGCTTCACCGAC 946                              GlnAlaArgIleLysPheGluThrGluGlyValGlyPheThrAsp                                  240245250                                                                      TTTTCGGACCGCAAGAACCGCGGCTATTTCGAATCCACCTATGTG991                               PheSerAs pArgLysAsnArgGlyTyrPheGluSerThrTyrVal                                 255260265                                                                      CGGACGCCGGGCGGCTGATGTTCGAAGCCACCCACAGCCTGGGCTTCACCCATG1045                     ArgThrProGlyGly                                                                270                                                                            ACGA GGATGAACGCTCGCTGGGCATGGACCTGAAGGTGTCGCCGCAGTTCGATGACAAGA1105              AGCA1109                                                                       (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2516 base pairs                                                    (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Double- stranded                                             (D) TOPOLOGY: Linear                                                           (ii) MOLECULE TYPE: Genomic DNA                                                (A) DESCRIPTION:                                                               (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: No                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Flavobacterium sp. Strain ATCC 39723                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       CCTTATGCGGCCGGCACGCAGGCCTGGTCTGTGCGT GAACCGGGCGGCTTATAGGCT57                   CCCATGGTTTAACTTTGCAACCATAATGTCGCCGGGCGTTACCTTTGCGCGATCGCCCGC117                CCGCAAGACCGATTCCGCCGCAAGCCGCGCTCATTTTCCGGCGCAGGTTATTCAGGTTCC177                GAATGCCCCAATTCGA CCGCACCGCCTATCGACGTTAACATCCGCGCCGGGCTTTGATAC237               CCGGCAAAAGCAAATGACGGCCAAGTTCATTATGAACAGGCACGTTATTTGGCAGAATCC297                AAAGTATTCATCGGGAGAGAGATTGTTATTATGTCGACCTATCCAATCAATGCG3 51                     MetSerThrTyrProIleAsnAla                                                       15                                                                             CCGGGCCAATCCGCCGATGCCGCGGTTTTGATCGTCGGCGGCGGGCCG399                            ProGlyGlnSerAlaAspAlaAlaValLeuIleValGlyGlyGlyPro                               10 1520                                                                        ACGGGGCTGATTGCGGCCAATGAATTGCTGCGCCGCGGCGTATCGTGC447                            ThrGlyLeuIleAlaAlaAsnGluLeuLeuArgArgGlyValSerCys                               2530 3540                                                                      CGCATGATCGATCGCCTGCCGGTCGCTCACCAGACGTCCAAATCCTGC495                            ArgMetIleAspArgLeuProValAlaHisGlnThrSerLysSerCys                               45 5055                                                                        ACCATCCATGCAAGATCGATGGAGATGATGGAACATATCGGCATCGCC543                            ThrIleHisAlaArgSerMetGluMetMetGluHisIleGlyIleAla                               6065 70                                                                        GCCCGCTACATAGAAACGGGCGTCAGGAGCAACGGGTTCACGTTCAAC591                            AlaArgTyrIleGluThrGlyValArgSerAsnGlyPheThrPheAsn                               758085                                                                         TTCGAG AATACGGATGCGAACGCGCTGCTCGACTTTTCCGTCCTGCCG639                           PheGlyAsnThrAspAlaAsnAlaLeuLeuAspPheSerValLeuPro                               9095100                                                                        GGCAGATATCCGTTCATCA CCATCTATAACCAGAATGAAACCGAACGG687                           GlyArgTyrProPheIleThrIleTyrAsnGlnAsnGluThrGluArg                               105110115120                                                                   GTGCTGCGGCACGATC TGGAGGCGACCTACAGCTTCCAGCCGGAATGG735                           ValLeuArgHisAspLeuGluAlaThrTyrSerPheGlnProGluTrp                               125130135                                                                      GGCACGCAGTTGCTGGCGCTCAATCAGG ATGAAAACGGCATCCGGGCT783                           GlyThrGlnLeuLeuAlaLeuAsnGlnAspGluAsnGlyIleArgAla                               140145150                                                                      GATCTGAGGCTGAAGGACGGGACGAAGCAGACGATCTCCC CGCGCTGG831                           AspLeuArgLeuLysAspGlyThrLysGlnThrIleSerProArgTrp                               155160165                                                                      GTGATCGGCGCGGACGGCGTGCGCAGCCGCGTCCGCGAATGCCTGGGC8 79                           ValIleGlyAlaAspGlyValArgSerArgValArgGluCysLeuGly                               170175180                                                                      ATCGCCTATGAAGGCGAGGATTATGAAGAAAATGTCCTTCAGATGATG927                            IleAlaTyr GluGlyGluAspTyrGluGluAsnValLeuGlnMetMet                              185190195200                                                                   GACGTCGGCATCCAGGATTTCGAAGCGGGCGACGACTGGATTCACTAT975                            AspVal GlyIleGlnAspPheGluAlaGlyAspAspTrpIleHisTyr                              205210215                                                                      TTCATCGGTCAGGACAAATTCGTCTTCGTGACGAAGCTGCCGGGTTCC1023                           PheIleGlyGlnAspLys PheValPheValThrLysLeuProGlySer                              220225230                                                                      AATTATCGCGTGATTATCAGCGACCTTGGCGGCGCCAACAAATCGAAT1071                           AsnTyrArgValIleIleSerAspLeuGly GlyAlaAsnLysSerAsn                              235240245                                                                      CTGGAAGAAACGCGGGAAGCCTTCCAGGGCTATCTCAGTTCCTTCGAC1119                           LeuGluGluThrArgGluAlaPheGlnGlyTyrLeuSerSer PheAsp                              250255260                                                                      GATCATGCGACGCTCGACGAGCCGCGTTGGGCGACCAAATGGCGGGTG1167                           AspHisAlaThrLeuAspGluProArgTrpAlaThrLysTrpArgVal                               265 270275280                                                                  TGGAAGCGGATGGCGACGGCCTATCGCAAGGGCAACGTCTTCCTGGCA1215                           TrpLysArgMetAlaThrAlaTyrArgLysGlyAsnValPheLeuAla                               285 290295                                                                     GGCGACGCGGCGCATTGCCATTCGCCGTCGGGCGGCAGCGGCATGAAC1263                           GlyAspAlaAlaHisCysHisSerProSerGlyGlySerGlyMetAsn                               300 305310                                                                     GTCGGCATGCAGGACGCCTTCAACCTGGGCTGGAAGATCGCCATGGTG1311                           ValGlyMetGlnAspAlaPheAsnLeuGlyTrpLysIleAlaMetVal                               315320 325                                                                     GAACGCGGCGAAGCCAAGCCCGACCTGCTCGACACCTATCATACCGAA1359                           GluArgGlyGluAlaLysProAspLeuLeuAspThrTyrHisThrGlu                               330335340                                                                      CGGA CGCCCGTCGCCCAGCAGTTGCTGGAAGGCACGCACGCCATGCAT1407                          ArgThrProValAlaGlnGlnLeuLeuGluGlyThrHisAlaMetHis                               345350355360                                                                   G AGATCATCATGGGGCATGGCAAGGGCCTGACCGACCGCATCGAATTG1455                          GluIleIleMetGlyHisGlyLysGlyLeuThrAspArgIleGluLeu                               365370375                                                                      ACGCAGGCGCCCG GTTGGCATGACGCCGCCACCTACCGCGTGTCGGGC1503                          ThrGlnAlaProGlyTrpHisAspAlaAlaThrTyrArgValSerGly                               380385390                                                                      ATGTCCTATAATTATCGCGACCAGC TCGTCAGCTTCAACGACGACCGG1551                          MetSerTyrAsnTyrArgAspGlnLeuValSerPheAsnAspAspArg                               395400405                                                                      CTGGCCGGACCCAGCGCTGGCGACCGCATTCCCGACG CGGAACTGGCG1599                          LeuAlaGlyProSerAlaGlyAspArgIleProAspAlaGluLeuAla                               410415420                                                                      CCCCGCATCCGGTTGTTCGACCTGGTCCGCAACACCCGGCCGACGCTG 1647                          ProArgIleArgLeuPheAspLeuValArgAsnThrArgProThrLeu                               425430435440                                                                   CTCGTGGCGCCCGCGACCGAAGCGGAAGTGGCGGAAGCGGAGAAGC TG1695                          LeuValAlaProAlaThrGluAlaGluValAlaGluAlaGluLysLeu                               445450455                                                                      CGCGACCTGATCCGCGAGCAGTGGCCGCTGGTGAAGCCCGTCCTCGTC1743                           Arg AspLeuIleArgGluGlnTrpProLeuValLysProValLeuVal                              460465470                                                                      CGTCCGCAGGGAAGCGAGGAATCCATCGAGGGCGACGTCCATGTCGAC1791                           ArgProGlnGlySer GluGluSerIleGluGlyAspValHisValAsp                              475480485                                                                      AGCTATGGCCAGCTCAAGCGCGAATGGGGCGACAATGCGAAGGGATGG1839                           SerTyrGlyGlnLeuLysArgGluTrp GlyAspAsnAlaLysGlyTrp                              490495500                                                                      GCGGCGCTGTTGAGGCCGGACAACTACATCCATGCGCGGGCGGGCCTG1887                           AlaAlaLeuLeuArgProAspAsnTyrIleHisAlaArg AlaGlyLeu                              505510515520                                                                   GATCGCGGCGATCTTCTGGTCCAGGCGATCGACGCGATGCTTGTGCCG1935                           AspArgGlyAspLeuLeuValGlnAlaIleAspAla MetLeuValPro                              525530535                                                                      TGCGCCTGAGGAGACCCGTGCGATGACAAACCCCGTTTCGACAATCGAC1984                          CysAla                                                                         539                                                                            ATGACGGTCACGCAGATCACCCGCGTGGCCAAGGACATCAACTCT TACGAACTTCGCCCG2044              GAACCCGGCGTGATATTGCCGGAGTTCACCGCGGGGGCGCATATCGGCGTTTCGCTTCCC2104               AACGGGATCCAGCGCACGTATTCGCTCGTCAACCCGCAGGCGAGAGGGACCGTTACGTGA2164               TCACGGTCAACCTCGACCGCAACAG CCGGGGCGGTCGCGCTACCTCCACGAGCAGTTGCG2224              GGTCGGGCAGCGCCTGTCCATCGTACCGCCCGCCAATAATTTCGCCCTGGTGGAGACAGC2284               CCCCCACTCCGTCCTGTTCGCGGGCGGCATCGGCATCACGCCGATCTGGTCGATGATCCA2344               ACGGT TGCGGGAACTCGGTTCCACCTGGGAGCTTCATTACGCCTGTCGCGGCAAGGATTT2404              CGTCGCCTACCGCCAGAACTGGAGCAGGCGGCGGCGGAGGCTGGAGCGAGATCACTCACT2464               CGATGAGAGCGACCGAAATTCTGACTGCGGCCGTGGCCAGGCGCAGAACGAC 2516                      (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1432 base pairs                                                    (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Double- stranded                                             (D) TOPOLOGY: Linear                                                           (ii) MOLECULE TYPE: Genomic DNA                                                (A) DESCRIPTION:                                                               (iii) HYPOTHETICAL: No                                                         (iv) ANTI-SENSE: NO                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                           (A) ORGANISM: Flavobacterium sp. Strain ATCC 39723                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       TAATGCACCGGATCGTGACGTATCCGAACCTGTTGCGGCTGTGGCCAAGCTGCCTCGCAT60                 GAAGCGTCGCGCCCACCATCAATCTCCGCCATGCCAAGGCCCATTATTACGGCAGCCATC120                CAAGCGTGAATCC CACCGGAATCGTCCCGTAGCCCCGCCCAACCTGCCGGGCCTAACCCT180               CCAGTCCTGACCAAAAAATCCTCCCCATCGAAAGATGGGGAGGGGGACCGTTCGCGCAGC240                GAATGGTGGAGGGGAACTGCGCAGGTCGCGCCATATTTCCTCCACTCCTCCCAAATACA T300               CCGACAACTGGAATCTGCTTATCCGATTTGCCCCATGGACTCTCCAGGGACGGAACGATA360                TGCTGCCTCGGCAAAATCGCTGAGGATATGAAGATGCCTGAAGTCAGTCTCTAC414                      MetAsnMetProGluValSerLeuTyr                                                    1 5                                                                            AATTACACCATGTCGATCTGTTCGATGAAGACCCGTCTGGCGATGGAG462                            AsnTyrThrMetSerIleCysSerMetLysThrArgLeuAlaMetGlu                               101520 25                                                                      GAATTCGGCGTCGACTATGACGACAAGCAGGTGGACATCGGCTTCGCC510                            GluPheGlyValAspTyrAspAspLysGlnValAspIleGlyPheAla                               3035 40                                                                        CTCGAAAACTTCGAACCCGACTATGTCCGGCTGAACGAAAAGGCGGTC558                            LeuGluAsnPheGluProAspTyrValArgLeuAsnGluLysAlaVal                               455055                                                                         GTGCCCAC GCTGGTCGTCGGCGACCGCGTCGTCACCAACAGCTACAAT606                           ValProThrLeuValValGlyAspArgValValThrAsnSerTyrAsn                               606570                                                                         ATCGTGCTGGAGGCCGCGAA CGTGGGCAAGGTCGGCATTCCCGCCGAC654                           IleValLeuGluAlaAlaAsnValGlyLysValGlyIleProAlaAsp                               758085                                                                         CCGGTCGAGAACAAGGCCGCGCTCGACTGGTT CCAGAAGGGCGACCAG702                           ProValGluAsnLysAlaAlaLeuAspTrpPheGlnLysGlyAspGln                               9095100105                                                                     GTCAATTTCCAGGTCATCACCTATGGGCA CAAGGGCGTGCCGCGCGGC750                           ValAsnPheGlnValIleThrTyrGlyHisLysGlyValProArgGly                               110115120                                                                      GACGAACTGCTGATCGCGCGCCGCGAACGCGCAAAGGAAT ATGCCGAA798                           AspGluLeuLeuIleAlaArgArgGluArgAlaLysGluTyrAlaGlu                               125130135                                                                      AAATATCCCGAACTGCGCTCCATCTACCAGGCCGCGCACGACCGCATC 846                           LysTyrProGluLeuArgSerIleTyrGlnAlaAlaHisAspArgIle                               140145150                                                                      GTGGAACATGGCAATTGCGCCTATGACGCGGACACGGTGGCCCAGGCG894                            ValGluH isGlyAsnCysAlaTyrAspAlaAspThrValAlaGlnAla                              155160165                                                                      GAAGTCGACCTGCAAAAGCGCCTCGACGAACTGGACGTGCATCTGGCG942                            GluValAspLeuGlnLys ArgLeuAspGluLeuAspValHisLeuAla                              170175180185                                                                   GACAAGCCGTTCATCGCGGGCAGCAACTACAGCATCGCCGACATCATG990                            AspLysProPheIle AlaGlySerAsnTyrSerIleAlaAspIleMet                              190195200                                                                      TGGACCGTCCTGCTGGCGCGGATCGAGATGCTCAACATGACGGCCTGG1038                           TrpThrValLeuLeuAlaArgIleGl uMetLeuAsnMetThrAlaTrp                              205210215                                                                      ATCAGCGAGCGCCCGAACCTGCTCGCTTATTATCAGCGGATGAAGGCG1086                           IleSerGluArgProAsnLeuLeuAlaTyrTyrGlnA rgMetLysAla                              220225230                                                                      CGCCGCAGCTTCGAAACGGCGCGGGTGATGCCCAATTGGAAGGGCGGC1134                           ArgArgSerPheGluThrAlaArgValMetProAsnTrpLysGlyGly                                235240245                                                                     ATCTGACCGTCGCCGGATCGGACCATTGCCAGGAAAAAAGGGAAAG1180                             Ile                                                                            250                                                                            GCGCGGGAAACCGCGCCCTTTCCCTTTTTGATGTTGCGGCCGACCGCAATGTGGCGATTA 1240              ATAGATATCCGGAAGATGGATAAGCCAATGAAAACACGGGTCGCGATGTAAAAGAAAATC1300               CGCAATGAAGATATTCGGCAATATTAGACACCTGTCCGATTTGCAGTCCAGCAACTGGAT1360               GTTGCCTATCCAATATGCGATTTGAAAAAAAACAATGCAG CGCTATTCTTCTTTCACTGC1420              TATTAAGAATTC1432                                                               (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 271 amino acid residues                                            (B) TYPE: Amino Acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: Linear                                                           (ii) MOLECULE TYPE: Protein                                                    (A) DESCRIPTION:Theoretical translation of open                                reading frame of pcpA gene                                                     (iii) HYPOTHETICAL:Yes                                                         (iv) ANTI-SENSE: No                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Flavobacterium sp. Strain ATCC 39723                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       MetGluThrAsnHisIleThrSerLeuHisH is                                             1510                                                                           IleThrIleCysThrGlyThrAlaGlnGlyAspIleAspPhePhe                                  152025                                                                         ValLysValMetGlyGlnArgPheValLysArgThrLe uPheTyr                                 303540                                                                         AspGlySerIleProIleTyrHisLeuTyrPheAlaAspGluLeu                                  455055                                                                         GlyThrProGlyThrValMetThrThrPh eProThrArgArgThr                                 606570                                                                         GlyGlnLysGlyArgLysGlySerAsnGlnPheThrValCysThr                                  758085                                                                         TyrAlaIleProLysGlySe rLeuGluTrpTrpIleGlyHisLeu                                 9095100                                                                        AsnAlaHisGlyIleAlaThrGlyGluProAlaProValSerAla                                  105110115                                                                      SerAlaMETT rpValSerSerIleArgLeuArgIleAspPheGlu                                 120125130                                                                      ValLeuGluAspGluAsnAspThrArgGlnProTyrAspSerPro                                  135140145                                                                       TyrValProIleGluHisAlaHisGlyGlyPheHisSerTrpThr                                 150155160                                                                      AlaSerValArgGluLeuGluAspMetAspPhePheMETGluAsn                                  165170 175                                                                     CysTrpAsnPheGluLysIleGlyGluGluGlyAsnArgHisArg                                  180185190                                                                      TyrArgValLysGlyThrThrGluSerGlyThrIleIleAspLeu                                  195 200205                                                                     LeuHisGluProAspArgArgGlnGlySerTrpThrIleAlaGlu                                  210215220                                                                      GlyIleIleHisHisGlyAlaPheAlaValProAspMetAspIle                                  225 230235                                                                     GlnAlaArgIleLysPheGluThrGluGlyValGlyPheThrAsp                                  240245250                                                                      PheSerAspArgLysAsnArgGlyTyrPheGluSerTh rTyrVal                                 255260265                                                                      ArgThrProGlyGly                                                                270                                                                            (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 538 amino acid residues                                            (B) TYPE:Amino Acid                                                            (C) STRANDEDNESS:                                                              (D) TOPOLOGY: Linear                                                           (ii) MOLECULE TYPE: Protein                                                     (A) DESCRIPTION:Theoretical translation of open                               reading frame of pcpB gene                                                     (iii) HYPOTHETICAL:Yes                                                         (iv) ANTI-SENSE: No                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Flavobacterium sp. Strain ATCC 39723                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       MetSerThrTyrProIleAsnAla                                                       15                                                                             ProGlyGln SerAlaAspAlaAlaValLeuIleValGlyGlyGlyPro                              101520                                                                         ThrGlyLeuIleAlaAlaAsnGluLeuLeuArgArgGlyValSerCys                               2530 3540                                                                      ArgMetIleAspArgLeuProValAlaHisGlnThrSerLysSerCys                               455055                                                                         ThrIleHisAlaArgSerMetGluMetMetGluHisIleGlyIleAl a                              606570                                                                         AlaArgTyrIleGluThrGlyValArgSerAsnGlyPheThrPheAsn                               758085                                                                         PheGlyAsnThrAspAlaAsnAlaLeuLeuAs pPheSerValLeuPro                              9095100                                                                        GlyArgTyrProPheIleThrIleTyrAsnGlnAsnGluThrGluArg                               105110115120                                                                   V alLeuArgHisAspLeuGluAlaThrTyrSerPheGlnProGluTrp                              125130135                                                                      GlyThrGlnLeuLeuAlaLeuAsnGlnAspGluAsnGlyIleArgAla                               140145 150                                                                     AspLeuArgLeuLysAspGlyThrLysGlnThrIleSerProArgTrp                               155160165                                                                      ValIleGlyAlaAspGlyValArgSerArgValArgGluCysLeuGly                               170 175180                                                                     IleAlaTyrGluGlyGluAspTyrGluGluAsnValLeuGlnMetMet                               185190195200                                                                   AspValGlyIleGlnAspPh eGluAlaGlyAspAspTrpIleHisTyr                              205210215                                                                      PheIleGlyGlnAspLysPheValPheValThrLysLeuProGlySer                               220225230                                                                      Asn TyrArgValIleIleSerAspLeuGlyGlyAlaAsnLysSerAsn                              235240245                                                                      LeuGluGluThrArgGluAlaPheGlnGlyTyrLeuSerSerPheAsp                               250255 260                                                                     AspHisAlaThrLeuAspGluProArgTrpAlaThrLysTrpArgVal                               265270275280                                                                   TrpLysArgMetAlaThrAlaTyrArgLysGlyAsnVal PheLeuAla                              285290295                                                                      GlyAspAlaAlaHisCysHisSerProSerGlyGlySerGlyMetAsn                               300305310                                                                      ValGlyMetGlnAspAlaPheA snLeuGlyTrpLysIleAlaMetVal                              315320325                                                                      GluArgGlyGluAlaLysProAspLeuLeuAspThrTyrHisThrGlu                               330335340                                                                      ArgThr ProValAlaGlnGlnLeuLeuGluGlyThrHisAlaMetHis                              345350355360                                                                   GluIleIleMetGlyHisGlyLysGlyLeuThrAspArgIleGluLeu                               365 370375                                                                     ThrGlnAlaProGlyTrpHisAspAlaAlaThrTyrArgValSerGly                               380385390                                                                      MetSerTyrAsnTyrArgAspGlnLeuValSerPheAsnAs pAspArg                              395400405                                                                      LeuAlaGlyProSerAlaGlyAspArgIleProAspAlaGluLeuAla                               410415420                                                                      ProArgIleArgLeuPheAspLeu ValArgAsnThrArgProThrLeu                              425430435440                                                                   LeuValAlaProAlaThrGluAlaGluValAlaGluAlaGluLysLeu                               445450 455                                                                     ArgAspLeuIleArgGluGlnTrpProLeuValLysProValLeuVal                               460465470                                                                      ArgProGlnGlySerGluGluSerIleGluGlyAspValHisValAsp                               475 480485                                                                     SerTyrGlyGlnLeuLysArgGluTrpGlyAspAsnAlaLysGlyTrp                               490495500                                                                      AlaAlaLeuLeuArgProAspAsnTyrIleHisAlaArgAlaG lyLeu                              505510515520                                                                   AspArgGlyAspLeuLeuValGlnAlaIleAspAlaMetLeuValPro                               525530535                                                                      CysAla                                                                         538                                                                             (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 250 amino acid residues                                            (B) TYPE: Amino Acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: Linear                                                           (ii) MOLECULE TYPE: Protein                                                    (A) DESCRIPTION:Theoretical translation of open                                reading frame of pcpC gene                                                     (iii) HYPOTHETICAL: Yes                                                        (iv) ANTI-SENSE: No                                                            (v) FRAGMENT TYPE:                                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Flavobacterium sp. Strain ATCC 39723                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       MetAsnMetProGluValSerLeuTyr                                                    15                                                                             AsnTyrThrMetSerIleCysSerMetLysThrArgLeuAlaMetGlu                               1015 2025                                                                      GluPheGlyValAspTyrAspAspLysGlnValAspIleGlyPheAla                               303540                                                                         LeuGluAsnPheGluProAspTyrValArgLeuAsnGl uLysAlaVal                              455055                                                                         ValProThrLeuValValGlyAspArgValValThrAsnSerTyrAsn                               606570                                                                         IleValLeuGluAlaAlaAsnVa lGlyLysValGlyIleProAlaAsp                              758085                                                                         ProValGluAsnLysAlaAlaLeuAspTrpPheGlnLysGlyAspGln                               9095100 105                                                                    ValAsnPheGlnValIleThrTyrGlyHisLysGlyValProArgGly                               110115120                                                                      AspGluLeuLeuIleAlaArgArgGluArgAlaLysGluTyrAlaGlu                               125 130135                                                                     LysTyrProGluLeuArgSerIleTyrGlnAlaAlaHisAspArgIle                               140145150                                                                      ValGluHisGlyAsnCysAlaTyrAspAlaAspThrValAlaG lnAla                              155160165                                                                      GluValAspLeuGlnLysArgLeuAspGluLeuAspValHisLeuAla                               170175180185                                                                   AspLysProPhe IleAlaGlySerAsnTyrSerIleAlaAspIleMet                              190195200                                                                      TrpThrValLeuLeuAlaArgIleGluMetLeuAsnMetThrAlaTrp                               205210 215                                                                     IleSerGluArgProAsnLeuLeuAlaTyrTyrGlnArgMetLysAla                               220225230                                                                      ArgArgSerPheGluThrAlaArgValMetProAsnTrpLysGlyGly                               235 240245                                                                     Ile                                                                            250                                                                            (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 base pairs                                                      (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Single- stranded                                             (D) TOPOLOGY: Linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       ATGGARACNAAYCAYATHAC 20                                                        (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 15 base pairs                                                      (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Single- stranded                                             (D) TOPOLOGY: Linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       DATRTCNCCYTGNGC15                                                              ( 2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 base pairs                                                      (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Single- stranded                                             (D) TOPOLOGY: Linear                                                           (x) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                        ACSTAYCCSATYAAYGCSCCSGGSCARWSSGCSGAYN37                                        (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acid residues                                             (B) TYPE: Amino Acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: Linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      ProGluValSerLeuTyrAsnTyrThrMetSerIleXaaSerMetLysThrArg                         151 015                                                                        (2) INFORMATION FOR SEQ ID NO: 11:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base pairs                                                      (B) TYPE: Nucleic Acid                                                         (C) STRANDEDNESS: Single- stranded                                             (D) TOPOLOGY: Linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      GATSSWCATSGTRTAGTTRTASAGSSWSACYTCSGG36                                     

We claim:
 1. A purified DNA molecule consisting essentially of a DNA sequence encoding pentachlorophenol hydroxylase (PcpB).
 2. A purified and isolated DNA molecule selected from the group consisting of:(a) a DNA molecule comprising nucleotide bases 328 to 1944 shown in SEQ ID NO. 2 or its complementary strand; and (b) DNA molecules of at least 20 nucleotides in length which hybridize under conditions of at least 75% stringency to the DNA molecules defined in (a).
 3. A purified and isolated DNA molecule encoding pentachlorophenol hydroxylase, wherein the DNA molecule hybridizes under conditions of at least 75% stringency with a DNA probe LX-6.
 4. A purified and isolated DNA molecule isolated from Flavobacterium sp. Strain ATCC 39723 and encoding pentachlorophenol hydroxylase, said DNA molecule comprising a DNA molecule selected from the group consisting of:(a) A 3.0 kb EcoRI DNA molecule isolated from the Flavobacterium sp. which DNA molecule hybridizes under conditions of at least 75% stringency with a DNA probe LX-6; and (b) DNA molecules of at least 20 nucleotides in length which hybridize under conditions of at least 75% stringency to the DNA molecules defined in (a).
 5. A transformed microorganism containing the DNA molecule of claim
 2. 6. A transformed microorganism containing a DNA molecule which encodes pentachlorophenol hydroxylase (PcpB).
 7. A purified DNA molecule consisting essentially of a DNA sequence encoding an enzyme that catalyzes the conversion of pentachlorophenol to 2,3,5,6-tetrachloro-p-hydroquinone.
 8. The purified and isolated DNA molecule according to claim 3 wherein the DNA molecule hybridizes under conditions of at least 85% stringency with the DNA probe LX-6.
 9. The purified and isolated DNA molecule of claim 2 wherein the DNA molecule hybridizes under conditions of at least 85% stringency to the DNA molecule defined in (a).
 10. The purified and isolated DNA molecule of claim 2 wherein the DNA molecule is at least 25 nucleotides in length.
 11. The purified and isolated DNA molecule of claim 9 wherein the DNA molecule is at least 25 nucleotides in length.
 12. A recombinant vector including a DNA molecule according to claim
 2. 13. A transformed microorganism containing a recombinant vector according to claim
 12. 14. A recombinant vector including a DNA molecule according to claim
 1. 15. A transformed microorganism containing a recombinant vector according to claim
 14. 